Quantitative sensors for phytohormone signalling systems

用于植物激素信号系统的定量传感器

• 批准号: BB/I023933/1
• 负责人: Richard Napier
• 金额: $ 14.1万
• 依托单位: University of Warwick
• 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%2FI023933%2F1
• 关键词: Quantitative; sensors; phytohormone; signalling; systems

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.

微生物传感器是非常小的探针，用于测量活组织中重要信号的浓度，并在真实的时间。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联，并且在传感器放置期间引起的创伤更少。为了有用，生物传感器需要对被测量的信号具有选择性并且高度敏感，以便记录活样品中这些信号的低浓度。沃里克电化学技术的进步最近允许开发一些神经递质的良好微生物传感器，这些神经递质对大脑功能很重要。一些植物激素与神经递质属于同一个化学信号家族。细胞分裂素是一种植物激素，其作用之一是帮助确定种子中贮藏组织的发育和大小。我们已经使用相同的沃里克制造技术的细胞分裂素的原型微生物传感器。植物中天然存在的一种酶，称为细胞分裂素脱氢酶（CKX），与细胞分裂素反应使其变性。这种酶已被纯化并包裹在涂层微电极上的可渗透玻璃层中。当浸入细胞分裂素时，随之产生的电信号既取决于细胞分裂素的存在，又取决于它们的浓度。这些原型生物传感器工作良好，但需要改进。这个项目是关于我们将如何测试其灵敏度的改进。我们的细胞分裂素微生物传感器可以通过使用具有更高活性的酶的替代形式来改进，所有这些都可以通过我们在捷克共和国奥洛穆茨的合作者获得。我们将测试不同的电极涂层，以改善连接性和测试方法，为电极提供纳米结构，以增加其表面积而不增加其尺寸。在优化了细胞分裂素生物传感器之后，我们将在植物样品上对其进行测试。最初，我们会把它浸在树液滴中，树液滴是在茎被切断根系时形成的。可以添加已知浓度的细胞分裂素以验证校准。更严格的测试将是将这些微传感器之一插入种子的存储组织中。在种皮上做一个微切口，我们将评估两种现成的ATP传感器在这种新环境中的性能，然后是细胞分裂素传感器，以记录激素信号强度随时间的实时变化，并将其与种子大小联系起来。我们从其他工作中了解到，高种子细胞分裂素产生更大的种子，通过了解信号如何以及何时到达，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多汁。我们将评估如何使这些生物传感器更加坚固，以便它们可以直接部署到植物中。我们将测试钨丝（比铂更强），从保护罩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-这是已知的植物细胞穿刺，包括单细胞测量。细胞分裂素微生物传感器的发展将是一个令人印象深刻的进步。基于ATP框架的坚固耐用的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填充，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，为当前和未来关于操纵植物种子大小和产量潜力的研究提供信息。其他传感器也将跟进。

项目成果

A highly selective biosensor with nanomolar sensitivity based on cytokinin dehydrogenase.

• DOI: 10.1371/journal.pone.0090877
• 发表时间: 2014
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Tian F;Greplová M;Frébort I;Dale N;Napier R
• 通讯作者: Napier R