PAPM-EAGER: Single-locus multi-hormone reporters for comprehensive plant phenotyping: a synthetic-biology approach.

PAPM-EAGER：用于综合植物表型分析的单位点多激素报告基因：一种合成生物学方法。

• 批准号: 1650139
• 负责人: Anna Stepanova
• 金额: $ 30万
• 依托单位: North Carolina State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-10-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1650139&HistoricalAwards=false
• 关键词: PAPM; EAGER; Single; locus; multi

All multicellular organisms rely on a set of hormones to control growth and development. These chemical compounds are produced by the organisms in extremely minute amounts, but are highly potent, capable of triggering dramatic physiological changes, and absolutely essential for proper development and defense against harmful environmental conditions or infections. In plants, there are nine classes of plant-produced chemical hormones. These control a wide-range of developmental processes, from seed germination to fruit ripening, and responses to the environment, from drought to herbivore attack. Despite the critical role of plant hormones, they are difficult to study, in part because they are hard to detect and quantify in order to know exactly when and where they are produced, transported and sensed. One way around this problem is to measure the specific biological effects triggered by the hormone instead of quantifying the hormone itself. Thus, growth regulators can be "visualized" with the help of hormone-responsive fluorescent reporter genes that make parts of the plants glow under UV light if the hormone is active in that part of the plant. This project seeks to produce a set of synthetic reporter genes that will make it possible to monitor the activity of multiple hormones at once, all in a single plant. Importantly, these reporters will be useful for the analysis of hormones in many plant species, including crop and ornamental plants and even trees. In addition to the obvious benefits to the plant research community highlighted above, this project is an ideal opportunity to introduce synthetic biology concepts and strategies to science undergraduates. Herein, the NCSU Biochemistry Undergraduate Research & Training Program (BURT-P) lead by the co-PI Dr. Ascencio-Ibanez (that currently houses 25 students) will be used as an effective platform to recruit and train up to 10 new students interested in synthetic biology. Furthermore, all personnel (the PIs, the postdoc and undergraduates) will take part in a previously established Plants4Kids bilingual outreach program for kids. Dr. Diego Orzaez, the developer of GoldenBraid gene assembly technology, will exchange materials, outreach ideas, and coordinate efforts for the popularization of gene assembly strategies among plant biologists.A major limitation of existing hormone reporters is their inability to monitor multiple hormones in a single plant line. Currently, the best available synthetic reporters (e.g., DR5:GUS, DR5v2-GFP, or DII-Venus for auxin and EBS:GUS for ethylene) need to be crossed or transformed into the background of interest (e.g., one's favorite mutant) individually due to the presence of the same selectable markers, identical reporter genes, and/or major silencing issues caused by the repetitive use of the same or overlapping DNA elements. If the plant community had access to multi-gene hormonal reporter constructs and transgenic lines, it would greatly facilitate the multi-faceted phenotypic analysis of pleiotropic mutants, as well as of the effects of stresses and other treatments that typically alter several hormonal pathways in parallel. Furthermore, visualizing the activity of several hormones simultaneously may help to uncover interactions and relationships between these growth regulators. For example, it is easy to imagine the tremendous impact simultaneous monitoring of the dynamics of all major hormones' activities with cellular resolution will have on our understanding of processes as important as drought, nutrient deficiency, plant-microbiome interactions, pathogen responses, or hormone crosstalk.

所有多细胞生物都依靠一组激素来控制生长和发育。这些化合物是由生物体以极度微量的生产产生的，但具有很高的效果，能够触发巨大的生理变化，对于适当的发育和防御有害环境条件或感染绝对至关重要。在植物中，有九类植物生产的化学激素。这些控制了从种子发芽到果实成熟的广泛发展过程，以及对环境的反应，从干旱到草食动物攻击。尽管植物激素的作用至关重要，但它们很难研究，部分原因是它们很难检测和量化，以便知道它们在何时何地产生，运输和感知。解决此问题的一种方法是测量激素触发的特定生物学作用，而不是量化激素本身。因此，如果激素在植物的那部分中活跃，则可以借助激素反应性荧光报告基因“可视化”生长调节剂，从而使植物的一部分在紫外线下发光。该项目旨在产生一组合成报告基因，这将有可能一次监测多种激素的活性，全部在单个工厂中。重要的是，这些记者将有助于分析许多植物物种的激素，包括作物和观赏植物，甚至树木。除了上面突出显示的植物研究社区的明显好处外，该项目是将合成生物学概念和策略引入科学本科生的理想机会。在此，由Co-Pi Ascencio-Ibanez博士领导的NCSU生物化学本科研究与培训计划（BURT-P）（目前拥有25名学生）将被用作招募和培训多达10名对合成生物学感兴趣的新学生的有效平台。此外，所有人员（PIS，博士后和本科生）将参加以前建立的植物4Kids双语外展计划。 Goldenbraid基因组装技术的开发商Diego Orzaez博士将交换材料，外展想和协调植物生物学家中基因组装策略的努力。现有的激素记者的主要限制是他们无法监测单个植物线中的多种激素。目前，最佳可用的合成记者（例如DR5：GUS，DR5V2-GFP或Dii-Venus用于生长素和EBS和EBS：ebs：GUS：for乙烯的GUS）需要跨或转变为感兴趣的背景（例如，由于相同的可选标记物，或者是相同的确定性，或者是同一选择的情况，或者是同一选择的情况下，或者是同一选择的情况，或者是Since sines，或者是Since since and Since and Since and Since and Since and Sine and Sine since and Sine and Sine since and since and since and Since and Since and Since and Since and Since and Sirce。重叠的DNA元素。如果植物群落可以进入多基因荷尔蒙报告构建体和转基因线，则将极大地促进对多效突变体的多方面表型分析，以及压力和其他治疗的影响，这些效果通常会在平行的平行中改变几种激素途径。此外，同时可视化几种激素的活性可能有助于发现这些生长调节剂之间的相互作用和关系。例如，很容易想象，同时对所有主要激素活性的动力学产生的巨大影响都将在我们对干旱，营养缺乏，植物 - 微生物组相互作用，病原体反应，病原体反应或激素串扰的过程中的理解中具有巨大的影响。

项目成果

From Ethylene-Auxin Interactions to Auxin Biosynthesis and Signal Integration

从乙烯-生长素相互作用到生长素生物合成和信号整合

• DOI: 10.1105/tpc.19.00339
• 发表时间: 2019
• 期刊: The Plant Cell
• 作者: Stepanova, Anna N.;Alonso, Jose M.
• 通讯作者: Alonso, Jose M.

Auxin catabolism unplugged: Role of IAA oxidation in auxin homeostasis

• DOI: 10.1073/pnas.1613506113
• 发表时间: 2016-09-27
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Stepanova, Anna N.;Alonso, Jose M.
• 通讯作者: Alonso, Jose M.

Translation regulation in plants: an interesting past, an exciting present and a promising future

• DOI: 10.1111/tpj.13520
• 发表时间: 2017-05-01
• 期刊: PLANT JOURNAL
• 影响因子: 7.2
• 作者: Merchante, Catharina;Stepanova, Anna N.;Alonso, Jose M.
• 通讯作者: Alonso, Jose M.

Monitoring Ethylene in Plants: Genetically Encoded Reporters and Biosensors

• DOI: 10.1002/smtd.201900260
• 发表时间: 2020-08-01
• 期刊: SMALL METHODS
• 影响因子: 12.4
• 作者: Fernandez-Moreno, Josefina-Patricia;Stepanova, Anna N.
• 通讯作者: Stepanova, Anna N.

An Improved Recombineering Toolset for Plants

• DOI: 10.1105/tpc.19.00431
• 发表时间: 2020-01-01
• 期刊: PLANT CELL
• 影响因子: 11.6
• 作者: Brumos, Javier;Zhao, Chengsong;Alonso, Jose M.
• 通讯作者: Alonso, Jose M.