We have made important progress in the direction of plant-based biosensors by combining the fungal bioluminescence pathway (FBP) with the promoter of heavy metal-responsive genes, and successfully designing a plant sensor that can detect soil heavy metals. This is a simple, efficient and low-cost plant-based biosensor, which makes it possible to quickly recognize feedback signals with the naked eye.

We utilized tobacco and periwinkle as plant-based biosensors, Tobacco (Nicotiana tabacum) is an annual herbaceous plant of the Solanaceae family that originated in tropical and subtropical America and is a rich source of the alkaloid nicotine, which has been used for a long time as an herbal medicine and social stimulant. So far, tobacco has been cultivated worldwide and it is not only a major cash crop but also a model plant for the study of plant genetics, molecular biology and cell biology. However, tobacco can only carry out leaf transient transformation experiments. In order to enrich the types of tissue organs in the plant chassis and give full play to the unique advantages of plants, we successfully tried and reproduced Agrobacterium-mediated transient transformation experiments by using the floral organs of the horticultural ornamental plant, periwinkle (Catharanthus roseus).

Firstly, we chose cadmium as the metal pollutant of interest, screened and verified the promoters of cadmium-specific expression genes in plants using various technical means (transcriptome screening, GUS staining verification and GFP fluorescence verification), and then transformed them into plant cells through vector construction, E. coli and Agrobacterium transformation, and finally expressed them successfully and verified their functions.

groups	NtNRAMP2	NtNRAMP5	NtNRAMP6
clean water
Cadmium (50ng/μL)
blank comparison
positive control
Conclusion	Strong response, strong specificity	Strongly responsive, weakly specific, this promoter may be responsive to drought in addition to cadmium	Strong response, strong specificity

After conducting experiments in multiple stages, we believe that in a limited time, we have screened out many very promising plant cadmium response regulatory switches, such as NtNRAMP2, NtNRAMP6, and NtNRAMP5.

Finally, the luminescence expression module was designed, which realized the expression of the FBP pathway and generated bioluminescence when exposed to cadmium metal, and successfully developed a set of simple and efficient biosensors for detecting heavy metal (e.g., cadmium) contamination in soil (water).

While our first focus is on cadmium metals, ideally our system could be applied to a variety of soil (water body) heavy metal contaminants. The system is based on a biosensor that creates a self-luminous system in the plant as well as promoters for responding to various metal contaminants.Exposure of the plant to the contaminated soil (water body) results in the triggering of the FBP pathway, which ultimately causes the plant to emit light that is visible to the naked eye, meaning that the plant can be quickly detected at night when it has been exposed to the contaminant. This is obvious to any observer and can be detected in time to take the necessary precautions.

Due to time constraints, we cannot continue to conduct more functional verification experiments for promoters.We requested help from the teachers at BGI. As the experimental difficulty of reconstructing the luminescent carrier itself is greater, we hope that they can conduct subsequent luminescence tests based on our existing experimental results. Finnally, our promoter PCR2 can really respond to cadmium.

Heavy metal contamination is a global problem that has serious implications for plant growth, food safety and environmental health. In order to engineer plant-based biosensors to exhibit bioluminescent properties in the presence of heavy metals, a novel and efficient method to detect and monitor heavy metal content in soil (water) was generated. We attempted to alter gene promoters to deliberately respond to external changes and successfully realized that the intensity of plant luminescence varies with the external heavy metal content, providing a solution for controlling the expression module.

Image source: (https://doi.org/10.1111/jipb.12892)

The GUS gene is present in some bacteria and encodes β-glucuronidase (GUS), a hydrolase that catalyzes the hydrolysis of many β-glucosidic esters. Because endogenous GUS activity does not exist in the vast majority of plant cells, the GUS gene is widely used as a reporter gene in transgenic plants, and is especially widely used in the study of spatiotemporal expression of genes. We utilized this technique to successfully overcome the challenge of a long plant growth cycle and complete the engineering closure in a limited time, for which a complete validation system was constructed, in which the developed plant parts, tools and protocols are exemplary for carrying out plant-related research.