Proof of Concept | Navarra_BG

This year we are doing a proof of concept to test if a plant could be used as a CO₂ biosensor. For that aim, we are testing high CO₂ inducible promoters of Arabidopsis thaliana to see which is better for future studies.

The main pitfall when you want to use plants as part of a science project is the time that you need to complete an experiment. For example, you need several months to get Arabidopsis seeds. Bacteria, by contrast, are able to replicate in 20 minutes. This is the reason why we developed a proof of concept for our project. This project will lay the groundwork for new projects that will design plants as CO₂ biosensors.

The promoters

A proof of concept is usually characterized by opening a new line of investigation. In our project, we are looking for promoters that could be induced by high levels of CO₂. There is a huge quantity of promoters in the genome of the Arabidopsis plants, so we had to make a previous selection of the promoters we going to test (See “Modeling” section).

*Arabidopsis thaliana*, the plant where we obtain our promoters

The way we tested if the promoters respond to high levels of CO₂ was by designing a transcriptional unit with the promoter, the GFP, and the NOS terminator. After that, we used a transient expression system in the model plant Nicotiana benthamiana, and then we grew them in two different conditions: one with ambient CO₂ and another one with high CO₂ concentration. Our rationale was that If the promoter was regulated by CO₂, the levels of GFP in the plants exposed to high CO₂ concentration should be higher than in plants exposed to ambient CO₂.

Our marker

The Green Fluorescent Protein (GFP) is our marker gene in this proof of concept. GFP is a gene that comes from the genome of a jellyfish, Aequorea victoria. The gene that codifies the GFP has been cloned and now is commonly used as a marker protein in synthetic biology. In our project, we use the GFP to analyze the level of expression of the Arabidopsis promoters under study.

*Aequorea victoria*, the jellyfish that has the GFP on its genome

Nicotiana benthamiana transformation

As we had to test the different selected promoters to see if they responded to high CO₂ levels, we transitory modified our plants.

Using this technique, we do not have to modify the seeds and wait until we can harvest and germinate them. We only transform the part of the plant that we infiltrate. The maximum expression of the marker gene used to be in the 3-5 days after the agroinfiltration.

Process of infiltration of *Agrobacterium tumefaciens* in *Nicotiana benthamiana*

Plants as biosensors

Once we have concluded our proof of concept, we could talk about the next steps of the project.

We aim to make plants that could be used as CO₂ biosensors. It is obvious that we need that the marker gene expression could be visible in simple sight, without the need of using a confocal microscope. To move from a proof of concept to a final project the next steps will be to replace GFP with a marker that produces in the plant an easily observable change and to transform the plants stably.

In addition, we feel that plants could be biosensors of a huge variety of molecules, which means that it could be possible to detect other types of substances such as CO, a toxic gas for human life, and also they could detect molecules of interest.

In conclusion, our proof of concept opens new lines of investigation such as using plants as biosensors to detect substances of interest in an economic and effective way.