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

所有多细胞生物都依赖一组激素来控制生长和发育。这些化合物由生物体产生，数量极少，但却非常有效，能够引发剧烈的生理变化，对于正常发育和防御有害环境条件或感染绝对至关重要。在植物中，有九类植物产生的化学激素。它们控制着广泛的发育过程，从种子发芽到果实成熟，以及对环境的反应，从干旱到食草动物的攻击。尽管植物激素发挥着关键作用，但它们很难研究，部分原因是它们很难检测和量化，以准确了解它们的生产、运输和感知的时间和地点。解决这个问题的一种方法是测量激素引发的特定生物效应，而不是量化激素本身。因此，生长调节剂可以在激素响应荧光报告基因的帮助下“可视化”，如果激素在植物的该部分活跃，则该基因会使植物的部分在紫外线下发光。该项目旨在生产一组合成报告基因，使同时监测单一植物中多种激素的活性成为可能。重要的是，这些报告基因可用于分析许多植物物种的激素，包括农作物和观赏植物，甚至树木。除了上面强调的对植物研究界的明显好处外，该项目还是向理科本科生介绍合成生物学概念和策略的理想机会。其中，由联合 PI 博士 Ascencio-Ibanez 领导的 NCSU 生物化学本科生研究与培训计划 (BURT-P)（目前有 25 名学生）将被用作一个有效的平台，招募和培训最多 10 名对合成生物学感兴趣的新生。此外，所有人员（PI、博士后和本科生）都将参加之前为儿童设立的 Plants4Kids 双语外展计划。 GoldenBraid基因组装技术的开发者Diego Orzaez博士将交流材料、推广想法并协调努力，以在植物生物学家中推广基因组装策略。现有激素报告器的一个主要限制是它们无法监测单个植物品系中的多种激素。目前，由于存在相同的选择性标记、相同的报告基因和/或由于重复使用相同或重叠的 DNA 元件而引起的主要沉默问题，最好的合成报告基因（例如，用于生长素的 DR5:GUS、DR5v2-GFP 或 DII-Venus 和用于乙烯的 EBS:GUS）需要单独杂交或转化到感兴趣的背景（例如，一个人最喜欢的突变体）中。如果植物群落能够获得多基因激素报告基因构建体和转基因品系，它将极大地促进多效性突变体的多方面表型分析，以及胁迫和其他通常同时改变多个激素途径的治疗的影响。此外，同时观察几种激素的活性可能有助于揭示这些生长调节剂之间的相互作用和关系。例如，很容易想象，通过细胞分辨率同时监测所有主要激素活动的动态将对我们理解干旱、营养缺乏、植物-微生物组相互作用、病原体反应或激素串扰等重要过程产生巨大影响。

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.