In planta imaging of pyridine nucleotides using second-generation fluorescent protein biosensors

Area of Science:

• Plant Biology
• Biochemistry
• Cellular Redox Homeostasis

Background:

• Pyridine nucleotide redox states are crucial for cellular metabolism and highly dynamic.
• Traditional biochemical methods for measuring pyridine nucleotides in plants are destructive and lack spatiotemporal resolution.
• Existing genetically encoded fluorescent biosensors for pyridine nucleotides suffer from pH sensitivity or slow dissociation rates, limiting their in vivo application.

Purpose of the Study:

• To develop and validate pH-stable genetically encoded fluorescent biosensors for real-time monitoring of pyridine nucleotide redox states in living plant cells.
• To generate transgenic Arabidopsis thaliana lines expressing these biosensors in specific cellular compartments (plastid stroma and cytosol).
• To enable spatiotemporal measurements of NADH/NAD+ ratio and NADPH levels in various plant tissues.

Main Methods:

• Utilized pH-stable genetically encoded fluorescent biosensors for pyridine nucleotides.
• Generated transgenic Arabidopsis lines expressing biosensors under the control of CaMV 35S (whole plant) and LAT52 (pollen tube) promoters.
• Visualized and quantified real-time redox dynamics in plastids and cytosol using fluorescent instruments.

Main Results:

• Successfully generated transgenic Arabidopsis lines expressing pH-stable pyridine nucleotide biosensors.
• Demonstrated the capability to monitor real-time dynamic changes in NADH/NAD+ ratio and NADPH levels in plastids and cytosol.
• Validated biosensor performance in various plant tissues, including pollen tubes, root hairs, and mesophyll cells.

Conclusions:

• The developed transgenic biosensor lines provide a powerful, non-destructive tool for studying plant redox biology.
• These tools overcome previous technological limitations, enabling accurate spatiotemporal measurements of pyridine nucleotide dynamics in planta.
• The findings are expected to significantly advance research in plant metabolic regulation and stress responses.