Cryo-scanning electron microscopy observations of vessel content during transpiration in walnut petioles. Facts or artifacts?

The current controversy about the "cohesion-tension" of water ascent in pla nts arises from the recent cryo-scanning electron microscopy (cryo-SEM) obs ervations of xylem vessels content by Canny and coworkers (1995). On the ba sis of these observations it has been claimed that vessels were emptying an d refilling during active transpiration in direct contradiction to the prev ious theory. In, this study we compared the cryo-SEM data with the standard hydraulic approach on walnut (Juglans regia) petioles. The results of the two techniques were in clear conflict and could not both be right. Cryo-SEM observations of walnut petioles frozen intact on the tree in a bath of liq uid nitrogen (LN2) suggested that vessel cavitation was occurring and rever sing itself on a diurnal basis. Up to 30% of the vessels were embolized at midday. In contrast, the percentage of loss of hydraulic conductance (PLC) of excised petiole segments remained close to 0% throughout the day. To fin d out which technique was erroneous we first analyzed the possibility that PLC values were rapidly returned to zero when the xylem pressures were rele ased. We used the centrifugal force to measure the xylem conductance of pet iole segments exposed to very negative pressures and established the releva nce of this technique. We then analyzed the possibility that vessels were b ecoming partially air-filled when exposed to LN,. Cryo-SEM observations of petiole segments frozen shortly after their xylem pressure was returned to atmospheric values agreed entirely with the PLC values. We confirmed, with water-filled capillary tubes exposed to a large centrifugal force, that it was not possible to freeze intact their content with LN2. We concluded that partially air-filled conduits were artifacts of the cryo-SEM technique in our study. We believe that the cryo-SEM observations published recently sho uld probably be reconsidered in the light of our results before they may be used as arguments against the cohesion-tension theory.