Quantitative sensors for phytohormone signalling systems

微生物传感器是一种非常小的探测器，用于实时测量活组织中重要信号的浓度。它们的小尺寸是有利的，因为收集的数据可以与更精确的组织放置相关，并且在传感器放置过程中造成的创伤更小。为了有用，生物传感器需要对被测量的信号具有选择性和高度的灵敏度，以便记录这些信号在活样本中的低浓度。华威大学电化学技术的进步最近使一些神经递质的良好微生物传感器得以开发，这些信号对大脑功能非常重要。一些植物激素与神经递质属于同一化学信号家族。细胞分裂素是一种植物激素，除其他作用外，还有助于决定种子中储存组织的发育和大小。我们已经使用相同的沃里克制造技术制作了细胞分裂素的微生物传感器原型。一种天然存在于植物中的酶，称为细胞分裂素脱氢酶(CKX)，与细胞分裂素反应，使其变性。该酶已被提纯，并被包裹在涂层微电极上的可渗透玻璃层中。当浸泡到细胞分裂素中时，随后的电信号取决于细胞分裂素的存在和它们的浓度。这些原型生物传感器工作得很好，但需要改进。这个项目是关于我们将如何测试他们敏感度的改善。我们的微生物细胞分裂素传感器可以通过使用活性更高的酶的替代形式来改进，这些都可以通过我们在捷克共和国奥洛穆克的合作者获得。我们将测试不同的电极涂层以改善连通性，并将测试方法赋予电极纳米结构，以在不增加其尺寸的情况下增加其表面积。在优化了细胞分裂素生物传感器之后，我们将在植物样本上对其进行测试。最初，我们会将其浸入树干液滴中，这些液滴是茎被切断根系统时形成的。可以添加已知浓度的细胞分裂素来验证校准。更严格的测试将是将这些微型传感器中的一个插入种子的储存组织中。通过在种皮上进行微切割，我们将评估现成的ATP传感器和细胞分裂素传感器在这一新环境中的性能，以记录激素信号强度随时间的实时变化，并将其与种子大小相关联。我们从其他工作中了解到，高种子细胞分裂素可以产生更大的种子，通过了解信号是如何以及何时到达的，我们可能能够为改善粮食安全的战略提供信息。并不是所有的植物组织都像种子贮藏组织那样柔软和多肉。我们将评估如何使这些生物传感器更坚固，以便它们可以直接部署到植物中。我们将测试钨丝(比铂更坚固)，从受保护的盖子中输送传感器尖端的微导管，并评估在玻璃微毛细管中制造传感器的前景-这是已知有利于植物细胞渗透的，包括单细胞测量。细胞分裂素微生物传感器的开发将是一个令人印象深刻的进步。基于成熟的ATP框架的加固生物传感器将说明这种生物传感器技术可以应用于解决植物生物学中的许多其他悬而未决的问题。我们的实验还将说明细胞分裂素如何驱动种子灌浆，以及我们如何将这些信息用于粮食安全。简而言之，生物传感器将提供所需的精确数据类型，为当前和未来有关操纵植物种子大小和产量潜力的研究提供信息。其他传感器将紧随其后。

Microbiosensors are very small probes used to measure the concentrations of important signals in living tissue and in real time. Their small size is advantageous because the data collected can be associated with more precise tissue placements and less trauma is induced during sensor placement. In order to be useful, biosensors need to be selective to the signal being measured and highly sensitive in order to record the low concentrations of these signals in living samples. Advances in electrochemistry technologies at Warwick have recently allowed the development of good microbiosensors for some neurotransmitters, signals important for brain function. Some plant hormones are part of the same family of chemical signals as the neurotransmitters. The cytokinins are phytohormones which, amongst other roles, help determine the development and size of the storage tissue in seeds. We have made a prototype microbiosensor for cytokinins using the same Warwick fabrication technologies. An enzyme that occurs naturally in plants known as cytokinin dehydrogenase (CKX) reacts with cytokinins to denature them. This enzyme has been purtified and encased in a permable glass layer on a coated microelectrode. When dipped into cytokinins the consequent electrical signal is dependent on both the presence of cytokinins and on their concentration. These prototype biosensors work well, but need improving. This project is about how we will test for improvements in their sensitivity. Our microbiosensors for cytokinin may be improved by using alternative forms of the enzyme with much higher activity, all available to us through our collaborators in Olomouc, Czech Republic. We will test different electrode coatings in order to improve connectivity and test methods to give nanostructure to the electrode to increase its surface area without increasing its size. Having optimised the cytokinin biosensor we will test it on plant samples. Initially we will dip it into sap droplets which form when the stem is cut off a root system. Known concentrations of cytokinin can be added to validate calibrations. A more exacting test will be to insert one of these microsensors into the storage tissue of a seed. A microincision into the seed coat will give access and we will evaluate the performance of both off-the-shelf ATP sensors in this new environment, and then the cytokinin sensors to record real-time changes in hormone signal strength with time and relate this to seed size. We know from other work that high seed cytokinins give bigger seeds, by understanding how and when the signal arrives we may be able to inform strategies for improving food security. Not all plant tissues are as soft and pulpy as seed storage tissue. We will evaluate how to make these biosensors more rugged so that they can be deployed directly into plants. We will test tungsten wires (stronger than platinum), microcatheters for delivering the sensor tip from a protected cover, and evaluate the prospects of making the sensors in glass micrcapillaries - which are known to be good for plant cell impalements, including single-cell measurements. Development of a microbiosensor for cytokinins will be a singularly impressive advance. Ruggedised biosensors based on the proven ATP framework will illustrate that this biosensor technology can be applied to address many other outstanding questions in plant biology. Our experiments will also illustrate how cytokinins drive seed filling and how we might use this information for food security. In short, the biosensors will provide the type of precision data needed to inform current and future studies on manipulating plant seed size and yield potential. Other sensors will follow.