Biogenesis of peptide signals for embryonic cuticle formation and pollen development

角质层是一种防止植物失水的疏水屏障，它是一项重要的创新，允许植物从原始的水环境过渡到干燥的陆地。种子发育过程中胚性角质层的形成受胚乳和胚乳之间双向信号通路的控制。该信号通路的中心元件是Twisted Seed 1(TWS1)，它是由胚胎产生和分泌的非活性前体。失活的多肽扩散到胚乳中，在那里被类枯草杆菌蛋白水解酶(SBT)ALE1处理和激活。然后，胚胎表皮表面的受体GSO1和GSO2感知到TWS1，激活的受体发出信号，继续产生角质层。一旦角质层完成，多肽就不能再通过屏障，信号就会关闭。因此，胚胎和胚乳之间的双向分子对话提供了可靠的自我调节质量控制机制，在萌发前保护角质层的完整性。除了由ALE1进行的C-端处理之外，激活TWS1还需要N-端处理。初步确定SBT1.8为N-端加工的候选酶。在这个项目中，我们将研究SBT1.8在TWS1生物发生中的作用，特别是关于它对底物识别的翻译后酪氨酸硫化的要求。底物选择性的决定因素将通过结构模型确定，并通过定点突变确认。我们将进一步分析SBT1.8在发育中的种子中的表达，并对功能丧失的突变体进行鉴定，以评估SBT1.8在体内对TWS1形成的贡献。TWS1属于Casparian条带完整性因子(CIF)多肽家族，包括CIF3和CIF4。CIF3和CIF4已被证实为GSO受体的新配体，但其生理功能尚不清楚。初步数据表明，CIF3和CIF4以及GSO受体参与了男性生殖发育所需的信号通路。本项目的第二部分着重于这一信号通路的特征，特别是在花药和花粉发育过程中激活CIF3和CIF4所需的蛋白酶的鉴定和特征。一种基于抑制剂的功能丧失分析方法将被用来识别在哪些组织中这些蛋白酶是活跃的，并且是CIF3和CIF成熟所必需的。在这些组织中表达的候选蛋白水解酶的特性将取决于它们在体外和体内切割和激活CIF3和CIF4多肽的能力。这些酶与CIF3和CIF4成熟的生理相关性将在遗传互补实验中进行评估。

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.