Unraveling the molecular circuits controlling plant cryptochrome activity

揭示控制植物隐花色素活性的分子回路

• 批准号: 402688457
• 负责人: Professor Dr. Alfred Batschauer
• 金额: --
• 依托单位: AG Molekulare Pflanzenphysiologie und Photobiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/402688457?language=en
• 关键词: Unraveling; molecular; circuits; controlling; plant

The physiological responses controlled by the cryptochrome (cry) blue light photoreceptors in plants are well studied and several of the downstream signaling components were identified. However, understanding the primary molecular mechanisms of how the light-induced activity of cryptochromes is achieved is still in its infancy. More recently it became evident that plant cryptochromes bind nucleotides such as ATP. Binding of these metabolites boosts the accumulation of the cryptochrome signaling state, and stabilizes the protein by inducing structural changes. In line with an important role of nucleotide binding for stimulating cryptochrome activity is the observation that a cry1 mutant (cry1L407F), which mimics the ATP-bound state, is constitutively active. The opposite process, inactivation of cryptochrome, is likewise not fully understood. It is hypothesized that re-oxidation through molecular oxygen of the semireduced FAD chromophore is the initial event in inactivation of cryptochrome. However, very recent data showed that plant cryptochromes dimerize upon photoactivation, that only the dimeric state is active, and that dimerization is inhibited by proteins named Blue light Inhibitors of Cryptochromes (BICs). We propose a regulatory circuit for controlling cryptochrome activity involving nucleotide and BIC binding to cryptochromes. This model will be tested in vitro using recombinant and purified cry wild type, hyperactive cry1L407F and BIC proteins to see how light, ATP and BICs affect the dimerization of crys. A mechanistic understanding of BIC function is facilitated by our finding that these proteins bind iron. We will investigate structural changes in cry1 caused by ATP, light and BIC-binding using hydrogen-deuterium exchange mass spectrometry and eventually X-ray crystallography. Finally, we will validate our in vitro data in planta by expressing our available non ATP-binding mutants of cry1 and cry2 in Arabidopsis and analyze their biological activity.

隐花色素蓝光光感受器在植物体内的生理反应已被广泛研究，并鉴定出了几种下游信号组分。然而，了解隐花色素的光诱导活性是如何实现的主要分子机制仍处于起步阶段。最近，植物隐花色素与核苷酸如ATP结合变得很明显。这些代谢物的结合促进了隐花色素信号状态的积累，并通过诱导结构变化来稳定蛋白质。与核苷酸结合刺激隐花色素活性的重要作用一致的是观察到模拟ATP结合状态的cry1突变体（cry1L407F）是组成型活性的。相反的过程，隐花色素的失活，同样没有完全理解。据推测，通过分子氧的半还原FAD发色团的再氧化是在隐花色素失活的初始事件。然而，最近的数据表明，植物隐花色素在光活化时二聚化，只有二聚体状态是活性的，并且二聚化被称为隐花色素蓝光抑制剂（BIC）的蛋白质抑制。我们提出了一个调控电路，用于控制隐花色素活性涉及核苷酸和BIC结合隐花色素。该模型将使用重组和纯化的cry野生型、高活性cry 1L 407 F和BIC蛋白进行体外测试，以了解光、ATP和BIC如何影响cry的二聚化。我们发现这些蛋白质结合铁促进了对BIC功能的机械理解。 我们将研究由ATP，光和BIC结合引起的cry1结构变化，使用氢氘交换质谱，并最终X射线晶体学。最后，我们将通过在拟南芥中表达我们现有的非ATP结合突变体cry1和cry2来验证我们在植物中的体外数据，并分析它们的生物学活性。