Development of plant-based hydrogen peroxide YFP nanosensors targeted to multiple sub-cellular locations

开发针对多个亚细胞位置的基于植物的过氧化氢 YFP 纳米传感器

• 批准号: BB/I020004/1
• 负责人: Nicholas Smirnoff
• 金额: $ 25.27万
• 依托单位: UNIVERSITY OF EXETER
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2011
• 资助国家: 英国
• 起止时间: 2011 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FI020004%2F1
• 关键词: Development; plant; based; hydrogen; peroxide

The oxygen (O2) we breathe is produced by plants when they photosynthesise. However, for cells that produce and/or consume O2 (by respiration) as a key part of their metabolism, there is an inherent danger and that is the production of reactive oxygen species (ROS). ROS arise as an inevitable consequence of O2 chemistry and if they accumulate, they cause oxidative damage to cell components (in particular chloroplasts and mitochondria) and can trigger the death of the cell. This is why plants make antioxidants, to limit the accumulation of ROS. One ROS, hydrogen peroxide (H2O2), is relatively stable while still being a powerful oxidant. H2O2 is used as a bleaching agent because of its powerful oxidising activity. H2O2 is made in plants as a bi-product of photosynthesis, respiration and many other chemical reactions that plant cells carry out. If it accumulates then, as with other ROS, it will cause oxidative damage. Evolution, though, has a habit of turning the potentially damaging into something useful. This is the case for H2O2. The accumulation of H2O2 in different parts of cells, before it attains damaging levels, acts to alter the expression of hundreds of genes by stimulating cellular signalling systems. H2O2 is an important cellular signalling molecule in bacteria, animal cells and especially plant cells. H2O2 stimulates cell signalling both internally and from cell-to-cell in response to many changes in the plant's environment, such as changes in light levels, wounding by herbivores and attack by pathogens. H2O2 is also used to regulate growth and development in plants, such as the development of secondary roots, the growth of pollen tubes and the hardening of cell walls. The intimate involvement of H2O2 in many aspects of plants' lives means it is imperative that we are able to locate and determine the changes in the level of H2O2 in different parts of the plant from the tissue down to the sub-cellular level. Until very recently this has not been possible. Knowing where, when and how much H2O2 accumulates is important in understanding if a plant is suffering oxidative damage or is actively signalling. The lack of technology for measuring H2O2 in real time, non-invasively and accurately means there are serious gaps in our understanding of how plants grow, reproduce and interact with their environment. Our aim is to provide the plant science community with means to locate and measure H2O2 at different sub-cellular locations in plant cells in real time. We can do this because a novel technology has been developed in which H2O2 can be specifically detected in cells using a genetically encoded protein sensor called HyPer. HyPer is a novel artificial protein which consists of a part (called a domain) of a bacterial protein called OxyR which changes shape when it specifically binds H2O2 .This OxyR domain is linked to a greatly modified fluorescent protein from a jellyfish, which changes its fluorescence characteristics in response to the change in shape of the OxyR domain. This fluorescence change, in response to H2O2, can be visualised by one of several types of microscope which allows the researcher to locate and measure changes in H2O2 concentration over time. HyPer has been shown to work in animal cells, bacteria and fish embryos. We have shown that HyPer works in exactly the same way in cells of roots and leaves of young seedlings. We aim to construct HyPer variants that will go to different locations in the cell so that researchers can build up a comprehensive picture of H2O2 accumulation in different tissues and conditions. However, HyPer expression is silent in older plants, which is common with other types of fluorescent sensors in plants. We have provided a number of solutions to this problem which will be deployed in this project to allow maximum and rapid uptake of this technology by the global plant science community, advancing knowledge of plant functions on a wide front.

我们呼吸的氧气（O2）是由植物光合作用产生的。然而，对于产生和/或消耗O2（通过呼吸）作为其代谢的关键部分的细胞，存在固有的危险，即活性氧（ROS）的产生。ROS作为O2化学的不可避免的结果出现，如果它们积累，它们会对细胞成分（特别是叶绿体和线粒体）造成氧化损伤，并可能引发细胞死亡。这就是为什么植物产生抗氧化剂，以限制ROS的积累。一种ROS，过氧化氢（H2 O2），是相对稳定的，同时仍然是一种强大的氧化剂。H2 O2由于其强大的氧化活性而被用作漂白剂。H2 O2在植物中作为光合作用、呼吸作用和植物细胞进行的许多其他化学反应的副产物而产生。如果它积累起来，就像其他ROS一样，它会引起氧化损伤。然而，进化有一个习惯，就是把潜在的破坏性变成有用的东西。这就是H2 O2的情况。H2 O2在细胞不同部位的积累，在达到破坏性水平之前，通过刺激细胞信号系统来改变数百个基因的表达。H2 O2是细菌、动物细胞，尤其是植物细胞中重要的细胞信号分子。H2 O2刺激细胞内部和细胞间的信号传导，以响应植物环境中的许多变化，例如光照水平的变化，食草动物的伤害和病原体的攻击。H2 O2还用于调节植物的生长和发育，如次生根的发育，花粉管的生长和细胞壁的硬化。H2 O2在植物生命的许多方面都有密切的参与，这意味着我们必须能够定位和确定植物不同部位从组织到亚细胞水平的H2 O2水平的变化。直到最近，这是不可能的。了解H2 O2在何处、何时以及积累了多少对于了解植物是否遭受氧化损伤或是否正在积极发出信号非常重要。缺乏真实的、非侵入性和准确地测量H2 O2的技术意味着我们对植物如何生长、繁殖以及与环境相互作用的理解存在严重差距。我们的目标是为植物科学界提供真实的实时定位和测量植物细胞中不同亚细胞位置的H2 O2的方法。我们可以做到这一点，因为已经开发了一种新技术，其中H2 O2可以使用一种名为Hyperer的基因编码蛋白传感器在细胞中特异性检测。Hyper是一种新型的人工蛋白质，它由一种称为OxyR的细菌蛋白质的一部分（称为结构域）组成，当它特异性地结合H2 O2时，该结构域会改变形状。该OxyR结构域与一种来自水母的经过极大修饰的荧光蛋白质连接，该荧光蛋白质响应于OxyR结构域形状的变化而改变其荧光特性。这种响应H2 O2的荧光变化可以通过几种类型的显微镜中的一种来观察，这使得研究人员能够定位和测量H2 O2浓度随时间的变化。Hyper已被证明在动物细胞、细菌和鱼类胚胎中起作用。我们已经证明，Hyper在幼苗的根和叶细胞中以完全相同的方式起作用。我们的目标是构建能够到达细胞不同位置的Hyper变体，以便研究人员能够全面了解H2 O2在不同组织和条件下的积累情况。然而，HyperPer表达在较老的植物中是沉默的，这与植物中其他类型的荧光传感器是常见的。我们已经为这个问题提供了一些解决方案，这些解决方案将在本项目中部署，以使全球植物科学界能够最大限度地快速吸收这项技术，从而在广泛的领域内推进植物功能的知识。

项目成果

Hydrogen Peroxide and Cell Signaling: Part B

过氧化氢和细胞信号传导：B 部分

• 发表时间: 2013
• 作者: Packer, Lester;Cadenas, Enrique
• 通讯作者: Cadenas, Enrique

Making open data work for plant scientists.

• DOI: 10.1093/jxb/ert273
• 发表时间: 2013-11
• 期刊: Journal of experimental botany
• 影响因子: 6.9
• 作者: Leonelli S;Smirnoff N;Moore J;Cook C;Bastow R
• 通讯作者: Bastow R

An update: improvements in imaging perfluorocarbon-mounted plant leaves with implications for studies of plant pathology, physiology, development and cell biology.

• DOI: 10.3389/fpls.2014.00140
• 发表时间: 2014
• 期刊: Frontiers in plant science
• 影响因子: 5.6
• 作者: Littlejohn GR;Mansfield JC;Christmas JT;Witterick E;Fricker MD;Grant MR;Smirnoff N;Everson RM;Moger J;Love J
• 通讯作者: Love J