The Dynamics of Embolism Repair in Xylem: In Vivo Visualizations Using High-Resolution Computed Tomography

Water moves through plants under tension and in a thermodynamically metastable state, leaving the nonliving vessels that transport this water vulnerable to blockage by gas embolisms. Failure to reestablish flow in embolized vessels can lead to systemic loss of hydraulic conductivity and ultimately death. Most plants have developed a mechanism to restore vessel functionality by refilling embolized vessels, but the details of this process in vessel networks under tension have remained unclear for decades. Here we present, to our knowledge, the first in vivo visualization and quantification of the refilling process for any species using high-resolution x-ray computed tomography. Successful vessel refilling in grapevine (Vitis vinifera) was dependent on water influx from surrounding living tissue at a rate of 6 × 10⁻⁴ μm s⁻¹, with individual droplets expanding over time, filling vessels, and forcing the dissolution of entrapped gas. Both filling and draining processes could be observed in the same vessel, indicating that successful refilling requires hydraulic isolation from tensions that would otherwise prevent embolism repair. Our study demonstrates that despite the presence of tensions in the bulk xylem, plants are able to restore hydraulic conductivity in the xylem.Vascular plants have evolved a simple but elegant system for long-distance transport of water and minerals through a network of nonliving, pipe-like cells. Whereas long-distance transport in animals is actively driven by positive pressure, most water transport in plants is passively driven by tension as explained by the Cohesion-Tension (C-T) theory (Dixon and Joly, 1894; Tyree, 2003). Water under tension is metastable however (Hayward, 1971), making the transport system inherently vulnerable to cavitation and blockage by gas embolisms (Tyree and Sperry, 1989). Direct measurements of negative pressures (tensions) in xylem (Wei et al., 1999) have confirmed the fundamental basis for the C-T theory of water transport in plants (e.g. Tyree, 2003), but many details regarding the susceptibility of the xylem network to cavitation and blockage by embolisms, and a thermodynamically plausible mechanism for the repair of these embolisms, remain unclear (Clearwater and Goldstein, 2005).Plants have apparently evolved mechanisms, including root pressure, to remove embolisms and restore water transport in vessels (Sperry et al., 1987; Tibbetts and Ewers, 2000; Isnard and Silk, 2009). Refilling of embolized vessels far from roots (Holbrook et al., 2001) and under a state of tension (Salleo and Gullo, 1986) is not well understood, but most hypotheses involve localized solute export into embolized vessels from adjacent living xylem parenchyma, osmotic movement of water into these vessels, and isolation of the refilling vessel from the tension in its local water environment (Tyree et al., 1999; Hacke and Sperry, 2003; Clearwater and Goldstein, 2005; Salleo et al., 2006). Embolism repair is complicated by the fact that xylem conduits (tracheids and vessels) form an interconnected network. While such a network will provide a low-resistance pathway for the bulk flow of water when the conduits are filled, if a cavitation event and subsequent embolism (gas bubble) either spontaneously occurs within a conduit, or spreads to it from another conduit, the presence of tension in this network should also quickly drain a conduit of its water and prevent its refilling. The spread of embolisms is limited by the small effective pore size of the connections between conduits (known as pit membranes), but under conditions of low plant water availability, embolisms do occur and spread (Tyree and Zimmermann, 2002; Choat et al., 2008), and evidence for the repair of embolized vessels, despite the presumed presence of a tension throughout the plant xylem, has been obtained in many species (Salleo et al., 1996; McCully et al., 1998; Zwieniecki and Holbrook, 1998; Kaufmann et al., 2009).A major limitation to the testing of these hypotheses and to our understanding of embolism repair has been the lack of in vivo observations at a sufficient resolution and an appropriate temporal scale to document how the refilling occurs. Here we present a new method for imaging the functional status of vessels using high-resolution x-ray computed tomography (HRCT), providing, to our knowledge, the first in vivo visualization of the refilling process for any species. Previous in vivo measurements of vessel refilling have been performed using NMR imaging, but the resolution was insufficient to determine the source of the refilling water (Holbrook et al., 2001; Scheenen et al., 2007). In vivo imaging at this scale allows for nondestructive visualization and measurement of the change in both air and water volume within the vessel lumen, giving unprecedented access to the mechanisms of embolism repair.