Understanding Plant Signaling via Innovations in Probe Delivery and Imaging
Jean T. Greenberg1* (firstname.lastname@example.org), Jessica M. Morgan1, Joanna Jelenska1, Dian Liu1, Gabriel Estrella dos Anjos1, Savannah Peralta1, Allison Rodas1, Jennifer L. Morrell-Falvey2, Chris Erb3, Bernard Abakah3, Martin Tindi3, Issaka Obuaba3, and Robert F. Standaert3
1The University of Chicago; 2Oak Ridge National Laboratory; and 3East Tennessee State University
The team aimed to optimize nanofibers to deliver DNA expression constructs to plant cells and to develop and use a custom-built fiber optic microscope and image analysis platform that enables iterative, non-destructive measurements of plant tissues over time. These tools were developed together with research aimed at understanding receptor-mediated trafficking of the growth-promoting PSK peptide and responses. The goals are (a) to use the microscope to image the trafficking of a fluorescent bioactive peptide and its receptor; (b) to improve and test different nanofiber designs for delivering probes to plants, (c) to further discover and validate the transcriptional changes due to PSK-induced signaling.
Microscope: The team upgraded the fiber optic microscope to optimize plant stabilization and imaging. The microscope includes 2 LED light sources plus a new white LED for brightfield imaging and interchangeable fiber optic lenses with different magnifications. The team created a mount to allow upright imaging, fabricated a 3D-printed leaf clip, and mounted the fiber on an extensible arm with 5-axis control (X-Y-Z plus pitch and yaw) for precise sample manipulation and fine focus to obtain high-resolution, iterative micrographs using live plants. With these refinements, the team observed receptor-dependent trafficking of the bioactive peptide PSK-TAMRA from one side of a leaf to the other.
Nanofibers: The team reported success in using vertically aligned carbon nanofiber arrays (VANCFs) to deliver and get expression of DNA constructs to various plant tissues (Morgan et al. 2022). Through a user proposal at the Center for Nanophase Material Sciences, the team designed and implemented a strategy to transfer VACNFs from a rigid silicon substrate to SU-8, a flexible substrate. Importantly, this permits use of the fibers to deliver reagents to curved plant structures. To overcome the hydrophobicity of SU-8, fibers in the flexible film were coated with a 2-3 nm layer of silicon oxide. Using a rolling motion to drive fibers through plant cells, the team succeeded in delivering DNA and dye to various curved plant organs. The team has been invited to submit a manuscript for JoVE Journal that includes the innovations that were recently achieved.
Biological materials/deliverables: The team conducted a time series transcriptomic analysis of root and shoot responses to PSK and revealed tissue-specific and time-dependent plant responses to this peptide hormone. The study also included a comprehensive analysis of PSK effects on whole seedlings during early development. The team found that PSK down-regulates the expression of a specific transcription factor family that regulates plant defense genes. This family is also the most enriched transcription factor in PSK down-regulated genes that associated with plant immunity. By comparing the transcriptome data with publicly available data, the team found that PSK has the opposite regulatory effects on that transcription factor family and defense-related genes compared with the responses triggered by the microbe-associated molecular pattern peptide flg22. This observation may explain the antagonism between these two peptide ligands (PSK and flg22). Researchers are currently testing predictions from the RNA-seq experiments using qPCR, physiological and biochemical readouts and are preparing a manuscript based on the findings. DOE funded research benefits for dissemination and deployment of bioimaging technology:
- A major advance is the iterative, non-destructive fluorescence imaging of bioactive peptides, their receptors and output signaling responses in intact plants that are highly relevant to improving traits for energy applications. This includes documenting changes in growth parameters and cell longevity and the accompanying signaling events.
- Nanofibers for introducing non-permeable probes and biomolecules into plant cells accelerates the discovery of plant signaling response components in many species in response to many stimuli/environmental Fibers serve the dual goal of providing fiducial markers for the iterative imaging developed. Finally, the approach can also be used for genome editing.
Morgan, J. M., et al. 2022. “An Efficient and Broadly Applicable Method for Transient Transformation of Plants using Vertically Aligned Carbon Nanofiber Arrays.” Frontiers in Plant Science 13. DOI: https://doi.org/10.3389/fpls.2022.1051340.
This research was supported by the DOE Office of Science, Office of Biological and Environmental Research (BER), grant no. DE-SC0019104.