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) 是植物进行光合作用时产生的。然而，对于产生和/或消耗 O2（通过呼吸）作为其代谢关键部分的细胞来说，存在一个固有的危险，那就是活性氧 (ROS) 的产生。 ROS 是 O2 化学反应不可避免的结果，如果它们积累，就会对细胞成分（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。这就是植物产生抗氧化剂来限制活性氧积累的原因。一种活性氧，即过氧化氢 (H2O2)，相对稳定，同时仍然是一种强大的氧化剂。 H2O2 由于其强大的氧化活性而被用作漂白剂。 H2O2 是植物中产生的，是植物细胞进行光合作用、呼吸作用和许多其他化学反应的副产品。如果它积累起来，就像其他活性氧一样，就会造成氧化损伤。然而，进化有一个习惯，就是把潜在的破坏性的东西变成有用的东西。 H2O2 就是这种情况。 H2O2 在细胞不同部位积累，在达到破坏性水平之前，会通过刺激细胞信号系统来改变数百个基因的表达。 H2O2 是细菌、动物细胞尤其是植物细胞中重要的细胞信号分子。 H2O2 刺激细胞内部和细胞间的信号传导，以响应植物环境的许多变化，例如光照水平的变化、食草动物的伤害和病原体的攻击。 H2O2还用于调节植物的生长和发育，例如次生根的发育、花粉管的生长和细胞壁的硬化。 H2O2 与植物生命的许多方面密切相关，这意味着我们必须能够定位并确定植物不同部位（从组织到亚细胞水平）H2O2 水平的变化。直到最近，这还是不可能的。了解 H2O2 积累的地点、时间和数量对于了解植物是否正在遭受氧化损伤或正在积极发出信号非常重要。缺乏实时、非侵入性且准确测量 H2O2 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实时定位和测量植物细胞中不同亚细胞位置的 H2O2 的方法。我们之所以能够做到这一点，是因为我们已经开发出一种新技术，可以使用一种名为 HyPer 的基因编码蛋白质传感器来特异性检测细胞中的 H2O2。 HyPer 是一种新型人工蛋白，由一种名为 OxyR 的细菌蛋白的一部分（称为结构域）组成，当它特异性结合 H2O2 时，该蛋白会改变形状。该 OxyR 结构域与来自水母的经过大幅修改的荧光蛋白相连，该蛋白会改变其荧光响应 OxyR 结构域形状变化的特征。这种响应 H2O2 的荧光变化可以通过几种类型的显微镜之一进行可视化，从而使研究人员能够定位和测量 H2O2 浓度随时间的变化。 HyPer 已被证明可以在动物细胞、细菌和鱼类胚胎中发挥作用。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发挥作用。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不同组织和条件下 H2O2 积累的情况。然而，HyPer 表达在较老的植物中是沉默的，这在植物中其他类型的荧光传感器中很常见。我们针对这个问题提供了许多解决方案，这些解决方案将在该项目中部署，以便全球植物科学界最大限度地、快速地采用这项技术，从而在更广泛的领域推进对植物功能的了解。

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