Biogenesis of peptide signals for embryonic cuticle formation and pollen development

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The cuticle, a hydrophobic barrier protecting plants from water loss, was an essential innovation allowing the transition of plants from their original, aqueous environment to dry land. Formation of the embryonic cuticle during seed development was shown to be controlled by a bi-directional signaling pathway for communication between the embryo and the endosperm. Central element of this signaling pathway is the peptide Twisted Seed 1 (TWS1) that is produced and secreted as an inactive precursor by the embryo. The inactive peptide diffuses into the endosperm, where it is processed and activated by the subtilisin-like protease (SBT) ALE1. TWS1 is then perceived by the receptors GSO1 and GSO2 at the epidermal surface of the embryo, and the activated receptors signal for continued cuticle production. As soon as the cuticle is complete, the peptide can no longer cross the barrier and signaling is shut off. The bi-directional molecular dialogue between the embryo and the endosperm thus provides a reliable self-regulatory quality control mechanism that safeguards cuticle integrity before germination. In addition to C-terminal processing by ALE1, N-terminal processing is required for activation of TWS1. Preliminary work identified SBT1.8 as a candidate protease for N-terminal processing. In this project we will investigate the role of SBT1.8 in the biogenesis of TWS1, particularly with respect to its requirement of post-translational tyrosine sulfation for substrate recognition. Determinants of substrate selectivity will be identified by structural modelling and confirmed by site-directed mutagenesis. We will further analyze the expression of SBT1.8 in developing seeds, and characterize loss-of-function mutants to assess the contribution of SBT1.8 to TWS1 formation in vivo. TWS1 belongs to the Casparian Strip Integrity Factor (CIF) family of peptides, including CIF3 and CIF4. CIF3 and CIF4 have been confirmed as novel ligands of the GSO receptors, but their physiological function is unknown. Preliminary data indicate that CIF3 and CIF4 as well as the GSO receptors are involved in a signaling pathway required for male reproductive development. The second part of this project focuses on the characterization of this signaling pathway, particularly on the identification and characterization of the proteases that are required for the activation of CIF3 and CIF4 during anther and pollen development. An inhibitor-based approach for loss-of-function analysis will be used to identify the tissues in which these proteases are active and required for CIF3 and CIF maturation. Candidate proteases expressed in these tissues will be characterized with respect to their ability to cleave and activate CIF3 and CIF4 peptides in vitro and in vivo. The physiological relevance of these proteases for CIF3 and CIF4 maturation will be assessed in genetic complementation experiments.