| Abstract: | Long-distance water transport through plant xylem is vulnerable to hydraulic dysfunction during periods of increased tension on the xylem sap, often coinciding with drought. While the effects of local and systemic embolism on plant water transport and physiology are well documented, the spatial patterns of embolism formation and spread are not well understood. Using a recently developed nondestructive diagnostic imaging tool, high-resolution x-ray computed tomography, we documented the dynamics of drought-induced embolism in grapevine (Vitis vinifera) plants in vivo, producing the first three-dimensional, high-resolution, time-lapse observations of embolism spread. Embolisms formed first in the vessels surrounding the pith at stem water potentials of approximately –1.2 megapascals in drought experiments. As stem water potential decreased, embolisms spread radially toward the epidermis within sectored vessel groupings via intervessel connections and conductive xylem relays, and infrequently (16 of 629 total connections) through lateral connections into adjacent vessel sectors. Theoretical loss of conductivity calculated from the high-resolution x-ray computed tomography images showed good agreement with previously published nuclear magnetic resonance imaging and hydraulic conductivity experiments also using grapevine. Overall, these data support a growing body of evidence that xylem organization is critically important to the isolation of drought-induced embolism spread and confirm that air seeding through the pit membranes is the principle mechanism of embolism spread.Water is transported through the xylem under tension and in a metastable state, making it inherently vulnerable to cavitation, the rapid phase change of liquid water to vapor (Dixon and Joly, 1895; Hayward, 1971; Tyree and Sperry, 1989). The resulting gas embolisms block water transport in the affected xylem vessel. It is widely accepted that embolisms spread between adjacent conduits when the pressure differential between gas-filled and water-filled conduits reaches a critical point where water vapor is aspirated through the pit membrane from the neighboring conduit (Tyree and Sperry, 1989; Tyree and Zimmermann, 2002). The resulting spread of embolisms through the xylem effectively reduces the hydraulic conductivity of the network, impairing the capacity to replace transpired water. The consequences of embolism formation can be dramatic, and it is now considered to be one of the major physiological factors driving reductions in forest primary productivity and drought-induced mortality in woody plants (Anderegg et al., 2012; Choat et al., 2012).Embolism spread between conduits is necessarily dependent on the number and orientation of the interconduit connections, but little is known about the organization of those connections or the spatial dynamics of embolism spread in vivo (Tyree and Zimmermann, 2002; Brodersen et al., 2010). This knowledge gap is largely due to the lack of a nondestructive visualization tool with sufficient resolution to study the propagation and spread of embolism. Previous efforts to visualize embolism in vivo utilized either cryo-scanning electron microscopy (cryo-SEM) or NMR imaging. Cryo-SEM yields fine resolution of frozen plant tissue, revealing the functional status of xylem conduits (i.e. water- or air-filled) at the time of freezing (Canny, 1997; Melcher et al., 2003; Cobb et al., 2007; Mayr et al., 2007; Johnson et al., 2012). Both transverse (Hukin et al., 2005; Sun et al., 2007; Johnson et al., 2012) and longitudinal (Utsumi et al., 1999) cryo-SEM sections have been prepared, but only provide a snapshot of a single point in time and in a single, two-dimensional plane. Similarly, NMR imaging was used in several studies as a nondestructive visualization tool to study the functional status of the xylem in vivo (Holbrook et al., 2001; Clearwater and Clark, 2003). However, the resulting images are typically of insufficient resolution to determine anything other than whether xylem conduits were filled with water or air. Three-dimensional (3D) imaging with NMR is challenging and is not frequently employed (Kuroda et al., 2006). Despite the availability of NMR, studies using this technology are largely focused to the spread of embolism over long periods of time (e.g. weeks Umebayashi et al., 2011] or months Pérez-Donoso et al., 2007]) rather than the short-term dynamics of embolism spread over the course of a few hours.Recently, high-resolution x-ray computed tomography (HRCT), a nondestructive diagnostic imaging tool, has been successfully used to study plant tissue in vivo (Brodersen et al., 2010, 2011). Synchrotron-based HRCT is based on the same principles as medical computed tomography systems but yields data with a spatial resolution of less than 5 µm and a temporal resolution of less than 30 min. Brodersen et al. (2011) expanded on this technology to study and map the 3D organization of grapevine (Vitis vinifera) stems and found that the functional status of the xylem could be determined in vivo. Brodersen et al. (2010) visualized the dynamics of embolism repair (i.e. the metabolically active refilling of embolized xylem conduits) in live plants using HRCT, including the growth of water droplets emerging from xylem parenchyma surrounding embolized vessels that eventually led to the dissolution of trapped gas inside the vessels. While we now have a better understanding of embolism repair and the physiological consequences of embolism spread are well documented (Tyree and Zimmermann, 2002; McDowell et al., 2008;
Cochard et al., 2009; Zwieniecki and Holbrook, 2009; Choat et al., 2012), the spatial dynamics and biophysics of embolism formation and spread in vivo have yet to be fully explored. Clearly, the spatial organization of xylem conduits plays a critical role in embolism repair and is likely even more influential in embolism spread, as direct connections between conduits are the most likely pathway through the network. Building on these findings and new techniques, we aimed to take advantage of HRCT imaging to provide the first high-resolution visualization of the spread of drought-induced embolism. |
