Understanding Plant Signaling via Innovations in Probe Delivery and Imaging

and the effects of plant stressors on the long distance trafficking of signaling ligands.

Progress in filling these knowledge gaps has been severely hindered by technical gaps in methods suitable for studying the relevant signaling mechanisms in vivo. This project will develop new technologies for the introduction and observation of signaling probes in live plants. Our approach will provide two key advances, the abilities (1) to deliver labeled non-membrane permeant probes, such as proteins, signaling reporters, and DNA, to plant tissue with single-cell precision and (2) to perform repeated imaging (e.g. fluorescence) of broad tissue areas within intact living plants, including leaves.

To realize the first advance (better probe delivery), arrays of carbon nanofiber spikes that transiently permeablize plant cells without causing substantial injury will be employed. To realize the second advance (repeated imaging), a novel imaging system will be developed that employs benign fiducial markers on plant surfaces for anatomical reference and robotically controlled fiber optic microscopes with CCD cameras to provide images of plant tissues and cells in situ. This imaging system will enable subcellular imaging. This system will provide reliable, non-invasive repeat imaging on intact plants and will be set up to image multiple plants in series with the goal of making the imaging automated.

These technical capabilities will be developed in the context of an important, but poorly understood, plant signaling system that controls biomass, cell growth, tissue longevity (senescence), and stress tolerance. This system consists of a secreted ligand called phytosulfokine (PSK), which is a 5 amino acid sulfated peptide, and its cell surface receptors (PSKRs). PSK signaling has both autonomous and non-autonomous effects. With the aid of the newly developed techniques, it will be possible to discern the trafficking patterns and abundance of the PSK ligand in the presence and absence of its receptors and abiotic stressors that affect growth and biomass. A key goal will be to measure the spatial and temporal signaling events at different scales (within treated cells and distal cells and tissues).

The ability to track signaling molecules in vivowill provide an unprecedented capability to explore the fundamental processes underlying plant growth and physiology, which in turn will accelerate the development of improved plant varieties in support of DOE missions. The plant systems to be analyzed include Arabidopsis and Populus. The former system will permit important benchmarks to be rapidly achieved, whereas the latter system, an important bioenergy crop, will show the potential of the imaging platform and probe delivery system to accelerate research findings.