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.
