Visualizing the effect of freezing on the vascular system of wheat in 3 dimensions by in-block imaging of dye-infiltrated plants

Authors: LIVINGSTON, DAVID; Tuong, Tan; Tisdale, Ripley; ZOBEL, RICHARD - NORTH CAROLINA STATE UNIVERSITY

Submitted to: American Journal of Botany
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/8/2022
Publication Date: 3/23/2022
Citation: Livingston, D.P., Tuong, T.D., Tisdale, R.H., Zobel, R. 2022. Visualizing the effect of freezing on the vascular system of wheat in 3 dimensions by in-block imaging of dye-infiltrated plants. American Journal of Botany. 2022:1-12. https://onlinelibrary.wiley.com/doi/full/10.1111/jmi.13101.
DOI: https://doi.org/10.1111/jmi.13101

Interpretive Summary: Infrared thermography has shown after roots of grasses freeze, ice spreads into the crown and then acropetally into leaves initially through vascular bundles. Leaves freeze singly with the oldest leaves freezing first and the youngest freezing later. Visualising the vascular system in its native 3-dimensional state will help in the understanding of this freezing process. A 2 cm section of the crown that had been infiltrated with aniline blue was embedded in paraffin and sectioned with a microtome. A photograph of the surface of the tissue in the paraffin block was taken after the microtome blade removed each 20 µm section. Two hundred to 300 images were imported into Adobe After Effects and a 3D volume of the region infiltrated by aniline blue dye was constructed. The reconstruction revealed that roots fed into what is functionally a region inside the crown that could act as a reservoir from which all the leaves are able to draw water. When a single root was fed dye solution, the entire region filled with dye and the vascular bundles of every leaf took up the dye; this indicated that the vascular system of roots was not paired with individual leaves. Fluorescence microscopy suggested the edge of the reservoir might be composed of phenolic compounds. When plants were frozen, the edges of the reservoir became leaky and dye solution spread into the mesophyll outside the reservoir. The significance of this change with regard to freezing tolerance is not known at this time.

Technical Abstract: A simple technique was developed for visualizing the vascular system of grasses in 3 dimensions by photographing the cut surface of a paraffin-embedded wheat plant that had been infiltrated with a dye solution. Three hundred images were imported into Adobe After Effects and reconstructed into a 3D volume of the vascular system using a previously documented technique. The new procedure described here eliminates the need to manually align images, which simplifies the reconstruction considerably. A large area within the crown was discovered that appears to act as a reservoir from which leaves draw water. To confirm this, a single root was used to draw up dye solution. This root filled the reservoir with dye and all leaves of the plant were able to pull dye solution to their tips. When the plant was frozen, this reservoir expanded almost 2-fold, presumably because the cells that contained the dye in unfrozen plants were damaged or altered by the freeze event. The significance of the change in the size of the reservoir after freezing is being investigated.