Biogenesis of peptide signals for embryonic cuticle formation and pollen development

胚胎角质层形成和花粉发育的肽信号的生物发生

• 批准号: 454320907
• 负责人: Professor Dr. Andreas Schaller
• 金额: --
• 依托单位: Fachgebiet Physiologie und Biochemie der Pflanzen (190c)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/454320907?language=en
• 关键词: Biogenesis; peptide; signals; embryonic; cuticle

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.

角质层是保护植物免受水分流失的疏水屏障，是一项重要的创新，使植物能够从原始的水环境过渡到干旱的陆地环境。在种子发育过程中，胚胎角质层的形成受到胚胎和胚乳之间双向信号通路的控制。该信号通路的核心要素是肽扭曲种子1 (TWS1)，它是由胚胎产生和分泌的非活性前体。无活性肽扩散到胚乳中，在那里它被枯草杆菌样蛋白酶（SBT） ALE1加工和激活。TWS1随后被胚胎表皮表面的受体GSO1和GSO2感知，激活的受体发出信号，继续角质层的产生。一旦角质层完成，肽就不能再穿过屏障，信号就被关闭了。因此，胚胎和胚乳之间的双向分子对话提供了一种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ALE1的c端加工外，TWS1的激活还需要n端加工。初步工作确定SBT1.8为n端加工的候选蛋白酶。在这个项目中，我们将研究SBT1.8在TWS1的生物发生中的作用，特别是关于其翻译后酪氨酸硫酸化对底物识别的要求。底物选择性的决定因素将通过结构建模来确定，并通过定点诱变来确认。我们将进一步分析SBT1.8在发育种子中的表达，并对功能缺失突变体进行表征，以评估SBT1.8对体内TWS1形成的贡献。TWS1属于Casparian Strip Integrity Factor （CIF）多肽家族，包括CIF3和CIF4。CIF3和CIF4已被证实是GSO受体的新型配体，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。本项目第二部分重点研究该信号通路的特征，特别是鉴定和表征花药和花粉发育过程中CIF3和CIF4激活所需的蛋白酶。一种基于抑制剂的功能丧失分析方法将用于识别这些蛋白酶活跃的组织，这些蛋白酶是CIF3和CIF成熟所必需的。在这些组织中表达的候选蛋白酶将根据其在体外和体内切割和激活CIF3和CIF4肽的能力进行表征。这些蛋白酶与CIF3和CIF4成熟的生理相关性将在遗传互补实验中进行评估。