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套激素来控制生长发育。这些化合物是由生物体产生的，数量极少，但却非常有效，能够引发剧烈的生理变化，对于正常发育和抵御有害环境条件或感染是绝对必要的。在植物中，有九种植物产生的化学激素。这些基因控制着广泛的发育过程，从种子萌发到果实成熟，以及对环境的反应，从干旱到食草动物的攻击。尽管植物激素起着至关重要的作用，但它们很难研究，部分原因是它们很难被检测和量化，从而无法确切地知道它们是在何时何地产生、运输和感知的。解决这个问题的一种方法是测量由激素触发的特定生物效应，而不是量化激素本身。因此，在激素反应荧光报告基因的帮助下，生长调节剂可以“可视化”，如果激素在植物的某个部分活跃，该基因会使植物的某些部分在紫外线下发光。该项目旨在产生一组合成报告基因，从而有可能同时监测单一植物中多种激素的活性。重要的是，这些报告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。除了上述对植物研究界的明显好处外，该项目也是向理科生介绍合成生物学概念和策略的理想机会。在此，NCSU生物化学本科生研究与培训计划（BURT-P）将被用作招募和培训10名对合成生物学感兴趣的新学生的有效平台，该计划由联合pi阿森西奥-伊巴涅斯博士领导（目前有25名学生）。此外，所有员工（pi，博士后和本科生）都将参加之前建立的Plants4Kids双语儿童推广计划。GoldenBraid基因组装技术的开发人员Diego Orzaez博士将在植物生物学家之间交换材料、拓展思想并协调推广基因组装策略的努力。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系中的多种激素。目前，最好的合成报告基因（如生长素的DR5:GUS、DR5v2-GFP或DII-Venus和乙烯的EBS:GUS）由于存在相同的可选择标记、相同的报告基因和/或重复使用相同或重叠的DNA元件引起的主要沉默问题，需要单独交叉或转化到感兴趣的背景（例如，一个人最喜欢的突变体）。如果植物群落能够获得多基因激素报告结构和转基因株系，将极大地促进对多效突变体的多方面表型分析，以及对胁迫和其他通常同时改变几种激素通路的处理的影响的分析。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，同时监测所有主要激素活动的动态与细胞分辨率将对我们理解干旱、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.