Modulation of Phytochrome B Signalling by Phosphorylation

通过磷酸化调节光敏色素 B 信号传导

• 批准号: BB/K006975/1
• 负责人: Ferenc Nagy
• 金额: $ 46.7万
• 依托单位: University of Edinburgh
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2013
• 资助国家: 英国
• 起止时间: 2013 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FK006975%2F1
• 关键词: Modulation; Phytochrome; Signalling; Phosphorylation

Plants are sessile organisms and therefore have to adapt their growth to changes in the environment. Among the abiotic and biotic environmental factors that regulate plant growth, light plays a distinguished role. Light is not only used to drive photosynthesis but it is also an important developmental clue to ensure optimal adaptation to the changing environment. To monitor variations in the wavelength, intensity, direction and duration of light, plants evolved a battery of photoreceptors. The photoreceptors active in red/far-red light are called phytochromes. Phytochromes are dimeric chromoproteins with one covalently linked tetrapyrrol chromophore per molecule, and they cycle between their biologically inactive (Pr = red absorbing) and active (Pfr = far-red absorbing) forms. It is the Pfr conformer which is imported into the nuclei and whose interaction with specific cellular factors is required to launch the signaling cascade. It follows that phytochrome signaling is quantitatively determined (i) by the number of Pfr molecules available and (ii) by the kinetics of protein-protein interactions between Pfr molecules and signal transducers.Phytochromes are often also referred to as light-regulated enzymes as it was shown that phytochrome-A (phyA) can autophosphorylate and phosphorylate other proteins in vitro and is phosphorylated by unknown kinase(s) in planta. These data were interpreted to mean that autophosphorylation reduces the amount of phytochrome Pfr by increasing its degradation whereas phosphorylation by an unknown kinase at other amino acid residues decreases the capacity of the Pfr form to bind to its authentic signaling partners. Thereby it is generally accepted that phosphorylation negatively regulates phyA controlled signaling and physiological responses. However, the topic is surrounded by considerable controversy as fundamental biochemical evidences are still missing to validate this theory. In this proposal, we outline a research program to elucidate how reversible phosphorylation regulates phytochrome-B (phyB) controlled responses at the molecular level. We show that phyB, the major photoreceptor regulating photomorphogenic responses in adult plants, is (i) phosphorylated at multiple sites in planta, (ii) this post-translational modification increases the rate of dark-reversion, a light independent conversion of the thermodynamically unstable Pfr to Pr form and thereby (iii) results in decreased responsiveness to red light. To test if phosphorylation also affects interaction of phyB with its downstream signalling partners, we will perform in vitro experiments to characterise binding of the mutated phyB proteins to Phytochrome Interacting Factor (PIF) proteins. We also intend to identify those phosphatases which dephosphorylate phyB. We will therefore express candidate phosphatase proteins in bacterial cells and treat phosphorylated phyB purified from plants with recombinant phosphatases purified from E.coli. Finally we will determine whether phyB indeed functions as a kinase. To this end, we will define if phosphorylation detected in planta can be recapitulated at least partly in vitro by characterising autophosphorylation of phyB Pr and Pfr purified from insect cells. If phyB indeed autophosphorylates in vitro, we will use this approach to identify the catalytic domain and the ATP binding sites essential for kinase activity of the photoreceptor. Taken together, these experiments will help us deciphering whether phyB is phopshorylated by itself or other yet unknown kinases or if both of these mechanisms are involved in mediating post-translational modification of the photoreceptor.

植物是固着生物，因此必须使其生长适应环境的变化。在调节植物生长的非生物和生物环境因素中，光起着显着的作用。光不仅用于驱动光合作用，而且还是确保最佳适应不断变化的环境的重要发育线索。为了监测光的波长、强度、方向和持续时间的变化，植物进化出了一组感光器。在红光/远红光下活跃的光感受器称为光敏色素。光敏色素是一种二聚体色素蛋白，每个分子有一个共价连接的四吡咯发色团，它们在生物非活性（Pr = 红光吸收）和活性（Pfr = 远红光吸收）形式之间循环。 Pfr 构象异构体被导入细胞核，需要与特定细胞因子相互作用才能启动信号级联反应。由此可见，光敏色素信号传导是通过 (i) 可用 Pfr 分子的数量和 (ii) Pfr 分子与信号转导器之间蛋白质-蛋白质相互作用的动力学来定量确定的。光敏色素通常也称为光调节酶，因为它研究表明光敏色素-A (phyA) 可以在体外自磷酸化和磷酸化其他蛋白质，并在植物中被未知激酶磷酸化。这些数据被解释为意味着自磷酸化通过增加其降解而减少光敏色素 Pfr 的量，而未知激酶在其他氨基酸残基处的磷酸化降低了 Pfr 形式与其真实信号传导伴侣结合的能力。因此，人们普遍认为磷酸化负向调节 phyA 控制的信号传导和生理反应。然而，由于仍然缺乏基本的生化证据来验证这一理论，因此该主题存在相当大的争议。在本提案中，我们概述了一项研究计划，以阐明可逆磷酸化如何在分子水平上调节光敏色素 B (phyB) 控制的反应。我们发现phyB是成年植物中调节光形态发生反应的主要光感受器，它（i）在植物的多个位点被磷酸化，（ii）这种翻译后修饰增加了暗回复率，这是一种热力学不稳定的光独立转换Pfr 形成 Pr 形式，从而 (iii) 导致对红光的响应性降低。为了测试磷酸化是否也会影响 phyB 与其下游信号传导伴侣的相互作用，我们将进行体外实验来表征突变的 phyB 蛋白与光敏色素相互作用因子 (PIF) 蛋白的结合。我们还打算鉴定那些使 phyB 去磷酸化的磷酸酶。因此，我们将在细菌细胞中表达候选磷酸酶蛋白，并用从大肠杆菌中纯化的重组磷酸酶处理从植物中纯化的磷酸化phyB。最后我们将确定 phyB 是否确实起到激酶的作用。为此，我们将通过表征从昆虫细胞中纯化的 phyB Pr 和 Pfr 的自磷酸化来定义是否可以在体外至少部分地重现植物中检测到的磷酸化。如果 phyB 确实在体外自动磷酸化，我们将使用这种方法来识别光感受器激酶活性所必需的催化结构域和 ATP 结合位点。总而言之，这些实验将帮助我们破译 phyB 是否被自身或其他未知激酶磷酸化，或者这两种机制是否都参与介导光感受器的翻译后修饰。

项目成果

New insights of red light-induced development.

红光诱导发育的新见解。

• DOI: 10.1111/pce.12880
• 发表时间: 2017-02-18
• 期刊: Plant, cell & environment
• 作者: András Viczián;Cornelia Klose;É. Ádám;F. Nagy
• 通讯作者: F. Nagy

Article

文章

• 发表时间: 2018
• 期刊: The Plant Journal
• 作者: Anita Hajdu

UV-B-responsive association of the Arabidopsis bZIP transcription factor ELONGATED HYPOCOTYL5 with target genes, including its own promoter.

拟南芥 bZIP 转录因子 ELONGATED HYPOCOTYL5 与靶基因（包括其自身启动子）的 UV-B 响应关联。

• DOI: http://dx.10.1105/tpc.114.130716
• 发表时间: 2014
• 期刊: The Plant cell
• 作者: Binkert M

Phosphorylation of phytochrome B inhibits light-induced signaling via accelerated dark reversion in Arabidopsis.

拟南芥中光敏色素 B 的磷酸化通过加速暗回复来抑制光诱导的信号传导。

• DOI: http://dx.10.1105/tpc.112.106898
• 发表时间: 2013
• 期刊: The Plant cell
• 作者: Medzihradszky M

High-level expression and phosphorylation of phytochrome B modulates flowering time in Arabidopsis.

光敏色素 B 的高水平表达和磷酸化可调节拟南芥的开花时间。

• DOI: http://dx.10.1111/tpj.12926
• 发表时间: 2015
• 期刊: for cell and molecular biology
• 作者: Hajdu A