Cryo-Scanning Electron Microscopy Observations of Vessel Content during Transpiration in Walnut Petioles. Facts or Artifacts?

Abstract

The current controversy about the "cohesion-tension" of water ascent in plants arises from the recent cryo-scanning electron microscopy (cryo-SEM) observations of xylem vessels content by Canny and coworkers (1995). On the basis of these observations it has been claimed that vessels were emptying and refilling during active transpiration in direct contradiction to the previous 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 liquid nitrogen (LN 2 ) suggested that vessel cavitation was occurring and reversing 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 find out which technique was erroneous we first analyzed the possibility that PLC values were rapidly returned to zero when the xylem pressures were released. We used the centrifugal force to measure the xylem conductance of petiole segments exposed to very negative pressures and established the relevance of this technique. We then analyzed the possibility that vessels were becoming partially air-filled when exposed to LN 2 . 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 LN 2 . 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 should probably be reconsidered in the light of our results before they may be used as arguments against the cohesion-tension theory.The "cohesion-tension" (CT) theory of sap ascent in plants was proposed more than a century ago by Bö hm (1893) and Dixon and Joly (1894). The theory postulates that (a) the xylem conduits form continuous water columns from the roots to the leaves, (b) the columns are held in place thanks to the capillary pressures that develop in the leaf mesophyll, and (c) leaf transpiration pulls water out of the xylem, which causes water absorption by the roots. A corollary of the theory is that high xylem tensions (negative pressures) must develop inside the xylem conduits. Over the past century a considerable amount of experimental data have been cumulated by plant physiologists, all consistent with the CT theory.However, recent direct measurements of sap pressure with xylem pressure probes (Zimmermann et al., 1994) and direct cryo-scanning electron microscopy (cryo-SEM) observations of xylem vessels content during transpiration (Canny, 1997b(Canny, , 1998b have questioned the validity of the CT theory. New experiments with the xylem pressure probe ...

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