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Scientists at Oak Ridge National Laboratory have developed the first-ever method of detecting ribonucleic acid, or RNA, inside plant cells using a technique that results in a visible fluorescent signal. The technology can help researchers detect and track changes in RNA and gene expression in real time, providing a powerful tool for the development of hardier bioenergy and food crops and for the detection of unwanted plant modifications, pathogens and pests.

RNA is a signaling molecule inside cells that is used to read the deoxyribonucleic, or DNA, code and convert it into functional parts such as proteins that are essential for plant growth and stress response. The ORNL-developed biosensor continuously monitors RNA levels in live plants, replacing a traditional destructive, time-consuming method used by scientists for collecting, processing and analyzing tissue.

"With this biosensor, scientists gain real-time insights into how cells reprogram themselves at a molecular level under changing environmental conditions such as drought or disease," said Xiaohan Yang, lead for the project at ORNL. The approach streamlines traditional methods used to verify gene expression in modified plants and can better detect plant physiology related to disease or nutrient stress, accelerating the development of better crops.

The biosensor involves splitting a ribozyme, an RNA molecule that can act as an enzyme to catalyze RNA splicing inside a cell, into two inactive pieces. Scientists at ORNL then attached the ribozyme pieces to guide RNA sequences designed to bind to a specific RNA target inside the plant cell.

When the guide RNA finds its target, the two ribozyme pieces reunite and become active. This triggers the assembly of a reporter protein that produces a visible fluorescence, revealing the RNA's location and abundance in the plant. The team's are detailed in the Plant Biotechnology Journal.

Scientists successfully demonstrated how the biosensor worked to detect a virus infecting a tobacco plant. When deployed in another plant, Arabidopsis, the biosensor revealed how genes are turned on and off inside the cells. The system can detect gene activity at different scales, from individual cells to the tissue level across the entire plant, including leaves, roots, flowers and stems.

"It's helpful to researchers to be able to see when and where a plant is starting to reprogram itself in response to conditions like drought," said Paul Abraham, co-author, bioanalytical chemist and manager of the DOE Secure Ecosystem Engineering and Design Science Focus Area, or SEED SFA, led by ORNL.

"We can then go in and precisely measure what's happening at the molecular level. With tools such as this, we can achieve a more complete understanding of what's happening at the cellular level and how that gets translated across the metabolic pathways throughout the plant."

Enabling real-time monitoring, new crop varieties

"The biosensor advances plant science in multiple ways," said Jerry Tuskan, co-author and director of the DOE Center for Bioenergy Innovation led by ORNL.

"Its versatility ranges from a fundamental science perspective of performing better functional genomics to practical use as a tool to screen plant performance for early detection of pathogens or other stress responses, even before those impacts result in outward changes to the plant."

ORNL plant scientists and synthetic biologists have achieved multiple breakthroughs for plant transformation, including the discovery of genes conferring drought tolerance and significantly boosting plant growth, the development of biosensors to detect CRISPR gene editing, and a gene-stacking technology to accelerate the development of new plant varieties. The work is aimed at innovations for domestic, affordable bio-based fuels, chemicals and materials, and continues on ORNL's long history of biological and genetics research.

"The discovery of messenger RNA originated with the work of ORNL biologists and chemists in the 1950s, using techniques developed during our work on the Manhattan Project," said Paul Langan, associate laboratory director for the Biological and Environmental Systems Science Directorate at ORNL.

"Today we continue to innovate in the molecular biology space. This new biosensing method developed by our scientists can track modifications to RNA, enabling better crops for both energy and food abundance."