Identifying which conduits are moving water in woody plants: a new HRCT-based method

In vivo imaging methods are useful for examination of plant vascular tissues, particularly in the identification of fluid vs gas-filled conduits; however, these methods may not allow for the simple identification of conductive conduits. Our aim in the present study was to develop a method that would allow for the in vivo identification of conductive conduits. Intact plants and segments of grapevine (Vitis vinifera L.) and intact American chestnut (Castanea dentata (Marshall) Borkh.) saplings were examined. We found that iohexol, a water soluble iodine-rich molecule, was a useful contrast agent. We also stained the xylem of segments and gasdried samples to compare between intact scans and excised segments. Iohexol could be readily fed through cut roots or stems into the transpiration stream, was successfully transported through the xylem and marked conductive vessels within highresolution computed tomography (HRCT) scans. Iohexol results were comparable to those obtained by staining cut segments, with iohexol detecting greater numbers of smaller conduits in some samples. Samples contained gas-filled conduits, as well as both conductive (containing iohexol tracer) and non-conductive (no iohexol tracer) fluid-filled vessels. Fluid-filled non-conductive vessels were likely still developing or were not connected to the sap stream by a low resistance pathway. We found minimal differences between intact and excised segments other than excision-related dilution of iohexol. Both vessels and vasicentric tracheids were filled with iohexol in chestnut, providing a new tool to study the functions of these different cell types. The use of iohexol as a tracer to identify conductive vessels may greatly improve the utility of HRCT as a tool in the study of plant hydraulic function. Future studies using HRCT will likely need to incorporate conductive vessel markers or controls into experiments due to the presence of non-conductive fluid-filled vessels within the xylem.