Carbon nanofiber arrays present a novel tool for biomolecule microdelivery to plants

Background/Question/Methods

Signaling within plants is essential for growth and development and is modulated by biotic and abiotic stresses within the environment. Understanding the role of signaling molecules in physiological processes is essential to developing novel strategies to promote plant health and immunity. Monitoring the movement of molecules within plants requires labeling with a detectable tag and delivery to the plant either by genetic transformation which works best in model plants or by physical methods such as root uptake or petiole-feeding and needs significant quantities of material. Microdelivery methods include microinjection via glass capillary or insect stylet and reduce the amount of material used but are technically challenging. We have developed a novel method for the microdelivery of labeled biomolecules to leaf tissue using vertically aligned carbon nanofiber (VACNF) arrays.  VACNFs are synthetic nanostructures similar in key dimensions (length, diameter and taper) to insect stylets. Individual fibers typically have tip diameters of ˂100 nm, base diameters of ~500 nm, and controllable lengths from a few µm to ~100 µm. Up to ~40,000 individual fibers are arrayed on a 2 X 2 mm chip. We used fluorescent and radiolabeled proteins to test the microdelivery potential of these arrays for Populus saplings.

Results/Conclusions

Our studies have shown that VACNFs are an effective tool for delivering molecules into Populus leaf epidermal cells with subsequent movement into the leaf vasculature and the plant proper. We demonstrated that VACNFs penetrate a single cell layer and effectively deliver sub microliter quantities of labeled proteins into epidermal cells without disrupting cell or tissue integrity or inducing a hydrogen peroxide mediated wound response. VACNF-delivered proteins moved quickly from the site of delivery into the plant vasculature. Fluorescence studies, using Oregon Green 488-labeled CmPP16-1, followed movement within a single leaf over a time period of minutes. Radiotracer studies, using ¹²⁵I-CmPP16-1 alone or mixed with a control protein, ¹³¹I-GFP, followed movement both locally and throughout the plant over time periods of minutes to hours. Both proteins moved bidirectionally, indicating phloem loading; however they exhibited different long distance trafficking patterns within individual plants. Combining VACNF delivery with radiolabeling overcomes issues of background, sensitivity and opacity that arise with fluorescence, thereby allowing long distance movement of small (fmol) amounts of protein to be followed in plants. This technology could be applied to elucidating the role of local and long-distance biomolecule transport in plant development, plant stress responses and plant microbe interactions.