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

我们呼吸的氧气（O2）是植物光合作用时产生的。然而，对于产生和/或消耗氧气（通过呼吸）作为其代谢的关键部分的细胞来说，存在固有的危险，那就是活性氧（ROS）的产生。活性氧是氧化学不可避免的结果，如果它们积累起来，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，以限制活性氧的积累。过氧化氢（H2O2）是一种相对稳定的活性氧，同时也是一种强大的氧化剂。H2O2因其强大的氧化活性而被用作漂白剂。H2O2是植物光合作用、呼吸作用和植物细胞进行的许多其他化学反应的副产物。如果它像其他活性氧一样积累起来，就会引起氧化损伤。然而，进化有一种习惯，就是把潜在的破坏性转化为有用的东西。这就是H2O2的情况。在达到有害水平之前，H2O2在细胞不同部位的积累通过刺激细胞信号系统改变了数百种基因的表达。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，以应对植物环境的许多变化，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发育，如次生根的发育、花粉管的生长和细胞壁的硬化。H2O2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H2O2水平的变化。直到最近，这还是不可能的。了解H2O2积聚的地点、时间和数量对于了解植物是否遭受氧化损伤或正在积极发出信号非常重要。缺乏实时、无创、准确测量H2O2的技术意味着我们对植物生长、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H2O2的方法。我们可以做到这一点，因为一项新技术已经开发出来，利用一种名为HyPer的基因编码蛋白质传感器，可以在细胞中特异性地检测到H2O2。HyPer是一种新型的人工蛋白质，它由一种叫做OxyR的细菌蛋白质的一部分（称为结构域）组成，当它特异性地与H2O2结合时，它会改变形状。这个OxyR结构域与来自水母的一种经过极大修饰的荧光蛋白相连，这种荧光蛋白会随着OxyR结构域形状的变化而改变其荧光特性。这种荧光变化，响应于H2O2，可以通过几种类型的显微镜之一可视化，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在幼苗的根和叶的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变异体，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，在较老的植物中，HyPer的表达是沉默的，这与植物中其他类型的荧光传感器一样。我们已经为这个问题提供了一些解决方案，这些解决方案将在这个项目中部署，以允许全球植物科学界最大限度地和快速地采用这项技术，在广泛的前沿推进植物功能的知识。

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.