Plant cell wall patterning and expansion mediated by protein-peptide-polysaccharide interaction

Assembly of cell wall polysaccharides into specific patterns is required for plant growth. A complex of RAPID ALKALINIZATION FACTOR 4 (RALF4) and its cell wall-anchored LEUCINE-RICH REPEAT EXTENSIN 8 (LRX8)-interacting protein is crucial for cell wall integrity during pollen tube growth, but its molecular connection with the cell wall is unknown. Here, we show that LRX8-RALF4 complexes adopt a heterotetrametric configuration in vivo, displaying a dendritic distribution. The LRX8-RALF4 complex specifically interacts with demethylesterified pectins in a charge-dependent manner through RALF4's polycationic surface. The LRX8-RALF4-pectin interaction exerts a condensing effect, patterning the cell wall's polymers into a reticulated network essential for wall integrity and expansion. Our work uncovers a dual structural and signaling role for RALF4 in pollen tube growth and in the assembly of complex extracellular polymers

Analyse fonctionnelle et génétique de nouvelles molécules de signalisation impliquées dans la régulation de la formation de la surface de l’embryon d’Arabidopsis thaliana

Seed development is a crucial step in Angiosperms life cycle. The seed is composed of three distinct compartments: (1) The testa, ensuring a protective function, (2) the endosperm, which plays a key nutritive role supporting (3) the embryo, the fate of which is to become the future plant. These three tissues develop concomitantly to form a viable seed. Such developmental coordination necessitates the involvement of communication between the compartments. In this context, I have studied genes involved in the establishment of the embryonic cuticle, a hydrophobic structure that surrounds the embryo, plays an essential post-germination function in regulating water loss and is thus critical for plant survival. At the beginning of my PhD, several proteins were known to be involved in the process of cuticle establishment, some of which were expressed in the endosperm and others in the embryo, hinting at the existence of molecular communication between the two tissues. On the endosperm side, the transcription factor ZOU controls the expression of ALE1, a subtilisin-like serine protease. On the embryo side, two receptors, GSO1 and GSO2, are involved. Genetic interaction between the genes encoding these proteins had confirmed their involvement the same signalling pathway. The molecular identities of these proteins led us to propose the existence of one or more unidentified peptides acting as messengers between the embryo and the endosperm. My research has allowed the characterization of novel proteins involved in the process of embryonic surface formation. The principal subject of my research has been CERBERUS, a peptide produced in the endosperm, the expression of which is controlled by ZOU, and which is necessary both for the formation of an intact embryonic cuticle and the production of a previously uncharacterised structure, the embryo sheath. I have demonstrated novel roles for GSO1 and GSO2 in embryo sheath deposition. Furthermore, I have generated preliminary data suggesting that a protein involved in peptide sulfation, TPST, is involved in the GSO1 GSO2 signalling pathway. Finally, I have shown that another protein involved in posttranslational protein modification, FRIABLE1 is involved in this same pathway. My results have advanced knowledge of the molecular mechanisms controlling embryonic surface formation in Arabidopsis.Le développement de la graine est une étape cruciale du cycle de vie des Angiospermes. La graine est composée de trois compartiments : (1) Le tégument, assurant un rôle protecteur, (2) l’albumen, qui a un rôle principalement nourricier pour (3) l’embryon, qui donnera la future plante. Ainsi, ces trois tissus se développent de concert pour former une graine viable. Une telle coordination présuppose que les différents compartiments communiquent entre eux. Dans ce contexte, j’ai étudié les gènes impliqués dans la formation de la cuticule embryonnaire, une structure hydrophobe recouvrant la plante et essentielle pour limiter les pertes d’eau, assurant ainsi sa survie. Au début de ma thèse, différents gènes étaient déjà connus, certains spécifiques de l’embryon, et d’autres spécifiques de l’albumen, renforçant l’idée de l’existence d’une communication moléculaire entre les deux tissus. Côté albumen, le facteur de transcription ZOU contrôle l’expression d’ALE1, une protéase. Côté embryon, deux récepteurs, GSO1 et GSO2, sont impliqués. L’étude de l’interaction génétique de ces différents gènes a permis de prouver leur appartenance à la même voie de signalisation. L’identité de ces gènes nous a amené à supposer l’existence d’un ou plusieurs peptides agissant comme messagers entre l’embryon et l’albumen. Ainsi, mes travaux de thèse ont permis de caractériser de nouveaux gènes impliqués dans ce processus, ainsi que certaines propriétés de la cuticule. Le principal est CERBERUS, dont l’expression est contrôlée par ZOU, un peptide sécrété par l’albumen qui est nécessaire pour la mise en place d’une cuticule fonctionnelle et la mise en place d’une structure non encore décrite à ce jour, la gaine embryonnaire. Un nouveau rôle pour GSO1 et GSO2 a aussi été démontré. Des résultats préliminaires suggèrent que TPST, une enzyme impliquées dans la sulfation des peptides, est impliquée dans la voie de signalisation étudiée. Enfin, mes travaux ont identifiés un autre gène, FRIABLE1, qui est aussi essentiel à la mise en place de la cuticule et joue dans la même voie de signalisation. Les découvertes associées à mes travaux de thèse ont permis de compléter et d’approfondir les connaissances sur les gènes impliqués dans la formation de la surface de l’embryon chez Arabidopsis

The EXTENSIN Enigma

International audienc

Structural basis for recognition of RALF peptides by LRX proteins during pollen tube growth

Plant reproduction relies on the highly regulated growth of the pollen tube for sperm delivery. This process is controlled by secreted RALF signaling peptides, which have previously been shown to be perceived by Catharanthus roseus RLK1-like (CrRLK1Ls) membrane receptor-kinases/LORELEI-like GLYCOLPHOSPHATIDYLINOSITOL (GPI)-ANCHORED PROTEINS (LLG) complexes, or by leucine-rich repeat (LRR) extensin proteins (LRXs). Here, we demonstrate that RALF peptides fold into bioactive, disulfide bond-stabilized proteins that bind the LRR domain of LRX proteins with low nanomolar affinity. Crystal structures of LRX2-RALF4 and LRX8-RALF4 complexes at 3.2- and 3.9-Å resolution, respectively, reveal a dimeric arrangement of LRX proteins, with each monomer binding one folded RALF peptide. Structure-based mutations targeting the LRX-RALF4 complex interface, or the RALF4 fold, reduce RALF4 binding to LRX8 in vitro and RALF4 function in growing pollen tubes. Mutants targeting the disulfide-bond stabilized LRX dimer interface fail to rescue lrx infertility phenotypes. Quantitative biochemical assays reveal that RALF4 binds LLGs and LRX cell-wall modules with drastically different binding affinities, and with distinct and mutually exclusive binding modes. Our biochemical, structural, and genetic analyses reveal a complex signaling network by which RALF ligands instruct different signaling proteins using distinct targeting mechanisms

Plant Surface Lipids and Epidermis Development

International audienceThe epidermis has a strategic position at the interface between the plant and the environment. In order to control exchanges with the environment as well as to protect the plant from external threats, the epidermis synthesises and secretes surface lipids to form a continuous, transparent and hydrophobic layer known as the cuticle. Cuticle formation is a strictly epidermal property in plants and all aerial epidermal cells produce some sort of cuticle on their surface. Conversely, all cuticularized plant surfaces are of epidermal origin. This seemingly anodyne observation has surprisingly profound implications in terms of understanding the function of the plant cuticle, since it underlies in part, the difficultly of functionally separating epidermal cell fate specification from cuticle biogenesis

Embryonic cuticle establishment: the great (apoplastic) divide

International audienceThe plant cuticle, a dynamic interface between plants and their environment, is formed by the secretion of hydrophobic lipids and waxes into the outer wall of aerial epidermal cells. Cuticle formation is such a ubiquitous feature of epidermal cells, and is of such fundamental importance for plant survival, that identifying and understanding specific developmental roles for this structure has been a major challenge for plant scientists. In recent work, we have tried to understand the functional relationships between a signaling feedback loop required for epidermal cell specification in developing plant embryos, and a seed specific signaling cascade, involving components localized both in the embryo and in the embryo surrounding endosperm, and necessary for embryo cuticle function. Analysis of the strongly synergistic genetic relationships between these 2 independent pathways, combined with mathematical simulations of the behavior of the signaling feedback loop, have allowed us to propose an important, and hitherto unsuspected, role for the embryonic cuticle as an apoplastic diffusion barrier, necessary for preventing the excessive diffusion of developmentally important signaling molecules away from developing embryo into surrounding tissues

Structural basis for recognition of RALF peptides by LRX proteins during pollen tube growth

Plant reproduction relies on the highly regulated growth of the pollen tube for proper sperm delivery. This process is controlled by secreted RALF signaling peptides, which have been previously shown to be perceived by CrRLK1Ls membrane receptor-kinases and leucine-rich (LRR) extensin proteins (LRXs). Here we demonstrate that RALF peptides are active as folded, disulfide bond-stabilized proteins, which can bind to the LRR domain of LRX proteins with nanomolar affinity. Crystal structures of the LRX-RALF signaling complexes reveal LRX proteins as constitutive dimers. The N-terminal LRR domain containing the RALF binding site is tightly linked to the extensin domain via a cysteine-rich tail. Our biochemical and structural work reveals a complex signaling network by which RALF ligands may instruct different signaling proteins – here CrRLK1Ls and LRXs – through structurally different binding modes to orchestrate cell wall remodeling in rapidly growing pollen tubes.SignificancePlant reproduction relies on proper pollen tube growth to reach the female tissue and release the sperm cells. This process is highly regulated by a family of secreted signaling peptides that are recognized by cell-wall monitoring proteins to enable plant fertilization. Here, we report the crystal structure of the LRX-RALF cell-wall complex and we demonstrate that RALF peptides are active as folded proteins. RALFs are autocrine signaling proteins able to instruct LRX cell-wall modules and CrRKL1L receptors, through structurally different binding modes to coordinate pollen tube integrity.</jats:sec

The Endosperm-Derived Embryo Sheath Is an Anti-adhesive Structure that Facilitates Cotyledon Emergence during Germination in Arabidopsis

International audienceGermination sensu stricto in Arabidopsis involves seed-coat and endosperm rupture by the emerging seedling root. Subsequently, the cotyledons emerge rapidly from the extra-embryonic tissues of the seed, allowing autotrophic seedling establishment [1, 2]. Seedling survival depends upon the presence of an intact seedling cuticle that prevents dehydration, which has hitherto been assumed to form the interface between the newly germinated seedling and its environment [- ]. Here, we show that in Arabidopsis, this is not the case. The primary interface between the emerging seedling and its environment is formed by an extra-cuticular endosperm-derived glycoprotein-rich structure called the sheath, which is maintained as a continuous layer at seedling surfaces during germination and becomes fragmented as cotyledons expand. Mutants lacking an endosperm-specific cysteine-rich peptide (KERBEROS [KRS]) show a complete loss of sheath production [6]. Although krs mutants have no defects in germination sensu stricto, they show delayed cotyledon emergence, a defect not observed in seedlings with defects in cuticle biosynthesis. Biophysical analyses reveal that the surfaces of wild-type cotyledons show minimal adhesion to silica beads in an aqueous environment at cotyledon emergence but that adhesion increases as cotyledons expand. In contrast, krs mutant cotyledons show enhanced adhesion at germination. Mutants with defects in cuticle biosynthesis, but no sheath defects, show a similar adhesion profile to wild-type seedlings at germination. We propose that the sheath reduces the adhesiveness of the cotyledon surface under the humid conditions necessary for seed germination and thus promotes seed-coat shedding and rapid seedling establishment