Craig R. Brodersen

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.

Measurement of vulnerability to water stress-induced cavitation in grapevine: a comparison of four techniques applied to a long-vesseled species

Among woody plants, grapevines are often described as highly vulnerable to water-stress induced cavitation with emboli forming at slight tensions. However, we found native embolism never exceeded 30% despite low xylem water potentials (Psi(x)) for stems of field grown vines. The discrepancy between native embolism measurements and those of previous reports led us to assess vulnerability curve generation using four separate methods and alterations (i.e. segment length and with/without flushing to remove embolism prior to measurement) of each. Centrifuge, dehydration and air-injection methods, which rely on measurement of percentage loss of hydraulic conductivity (PLC) in detached stems, were compared against non-invasive monitoring of xylem cavitation with nuclear magnetic resonance (NMR) imaging. Short segment air-injection and flushed centrifuge stems reached >90 PLC at Psi(x) of-0.5 and -1.5 MPa, respectively, whereas dehydration and long-segment air-injection measurements indicated no significant embolism at Psi(x) > -2.0 MPa. Observations from NMR agreed with the dehydration and long segment air-injection methods, showing the majority of vessels were still water-filled at Psi(x) > -1.5 MPa. Our findings show V. vinifera stems are far less vulnerable to water stress-induced cavitation than previously reported, and dehydration and long segment air-injection techniques are more appropriate for long-vesseled species and organs.

Maintenance of xylem network transport capacity: a review of embolism repair in vascular plants

Maintenance of long distance water transport in xylem is essential to plant health and productivity. Both biotic and abiotic environmental conditions lead to embolism formation within the xylem resulting in lost transport capacity and ultimately death. Plants exhibit a variety of strategies to either prevent or restore hydraulic capacity through cavitation resistance with specialized anatomy, replacement of compromised conduits with new growth, and a metabolically active embolism repair mechanism. In recent years, mounting evidence suggests that metabolically active cells surrounding the xylem conduits in some, but not all, species are capable of restoring hydraulic conductivity. This review summarizes our current understanding of the osmotically driven embolism repair mechanism, the known genetic and anatomical components related to embolism repair, rehydration pathways through the xylem, and the role of capacitance. Anatomical differences between functional plant groups may be one of the limiting factors that allow some plants to refill while others do not, but further investigations are necessary to fully understand this dynamic process. Finally, xylem networks should no longer be considered an assemblage of dead, empty conduits, but instead a metabolically active tissue finely tuned to respond to ever changing environmental cues.

In Vivo Visualizations of Drought-Induced Embolism Spread in Vitis vinifera

Time-lapse x-ray tomography uncovers the importance of intervessel connections in the xylem network in drought-induced embolism. 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.

Automated analysis of three‐dimensional xylem networks using high‐resolution computed tomography

Connections between xylem vessels represent important links in the vascular network, but the complexity of three-dimensional (3D) organization has been difficult to access. This study describes the development of a custom software package called TANAX (Tomography-derived Automated Network Analysis of Xylem) that automatically extracts vessel dimensions and the distribution of intervessel connections from high-resolution computed tomography scans of grapevine (Vitis vinifera) stems, although the method could be applied to other species. Manual and automated analyses of vessel networks yielded similar results, with the automated method generating orders of magnitude more data in a fraction of the time. In 4.5-mm-long internode sections, all vessels and all intervessel connections among 115 vessels were located, and the connections were analyzed for their radial distribution, orientation, and predicted shared wall area. Intervessel connections were more frequent in lateral than in dorsal/ventral zones. The TANAX-reconstructed network, in combination with commercial software, was used to visualize vessel networks in 3D. The 3D volume renderings of vessel networks were freely rotated for observation from any angle, and the 4.5 μm virtual serial sections were capable of being viewed in any plane, revealing aspects of vessel organization not possible with traditional serial sections.

Do changes in light direction affect absorption profiles in leaves

Leaf anatomy plays a functional role in propagating light through the leaf; palisade mesophyll has been shown to facilitate the channelling of collimated light deeper into the spongy mesophyll. Direct measurements of the propagation of diffuse light into the leaf, however, are absent. Using chlorophyll fluorescence imaging of leaf cross-sections, we measured light absorption profiles in leaves under direct (collimated), diffuse and low-angle monochromatic light. Low-angle and diffuse light was absorbed closer to the irradiated surface than direct light perpendicular to the surface. The shapes of internal absorption profiles indicated that leaves were influenced by the directional quality of the incident light. In addition, absorption profiles revealed that leaves were not simple light absorbing objects and that cellular anatomy influences the direction of light travelling into the mesophyll. These findings also suggest a mechanism for previously measured differences in leaf level photosynthesis under opposing light regimes.

Centrifuge technique consistently overestimates vulnerability to water stress‐induced cavitation in grapevines as confirmed with high‐resolution computed tomography

Vulnerability to cavitation is a key variable defining the limits to drought resistance in woody plants (Kursar et al., 2009). This trait is typically assessed by a vulnerability curve, which can be generated by a range of methods, including dehydration (Sperry et al., 1988), air injection (Cochard et al., 1992), and centrifugation (Alder et al., 1997). Results from two recent papers suggest that one of the most widely used methods, the centrifuge technique, overestimates vulnerability to cavitation in species with very long vessels (Choat et al., 2010; Cochard et al., 2010). Typically, the centrifuge technique produces characteristic ‘R shaped’ curves for long-vesseled species, compared with ‘S shaped’ curves produced by the dehydration method (Cochard et al., 2010). Both research groups proposed that open vessels contained in the centrifuged samples were responsible for this artifact. Grapevine (Vitis vinifera L.), a liana species known to have unusually long and wide vessels, appears to be particularly susceptible to artifacts with the centrifuge method (Choat et al., 2010), but the conclusions of this paper have been challenged by Jacobsen & Pratt (2012). They contend the dehydration technique actually underestimates vulnerability to cavitation in grapevine because the production of gels and ⁄ or tyloses causes a decline in maximum specific hydraulic conductivity (Ks max) over time. On the basis of their results and previously published hydraulic data, they concluded that the centrifuge technique is the most appropriate technique for estimating vulnerability to embolism (see details in Jacobsen & Pratt, 2012). Here we demonstrate that declining Ks max did not influence the results of Choat et al. (2010) and present new evidence from high-resolution computed tomography (HRCT) to support our original conclusions and refute those of Jacobsen & Pratt (2012). We also contend that the analysis of previous literature presented in Jacobsen & Pratt (2012) was oversimplified and obscured the specific comparison of cavitation resistance in current year shoots of grapevine. Overall, the findings presented in Jacobsen & Pratt (2012) for V. vinifera cv Glenora are in direct contrast to published and unpublished results generated by our research groups for other V. vinifera varieties.

Do epidermal lens cells facilitate the absorptance of diffuse light

Many understory plants rely on diffuse light for photosynthesis because direct light is usually scattered by upper canopy layers before it strikes the forest floor. There is a considerable gap in the literature concerning the interaction of direct and diffuse light with leaves. Some understory plants have well-developed lens-shaped epidermal cells, which have long been thought to increase the absorption of diffuse light. To assess the role of epidermal cell shape in capturing direct vs. diffuse light, we measured leaf reflectance and transmittance with an integrating sphere system using leaves with flat (Begonia erythrophylla, Citrus reticulata, and Ficus benjamina) and lens-shaped epidermal cells (B. bowerae, Colocasia esculenta, and Impatiens velvetea). In all species examined, more light was absorbed when leaves were irradiated with direct as opposed to diffuse light. When leaves were irradiated with diffuse light, more light was transmitted and more was reflected in both leaf types, resulting in absorptance values 2-3% lower than in leaves irradiated with direct light. These data suggest that lens-shaped epidermal cells do not aid the capture of diffuse light. Palisade and mesophyll cell anatomy and leaf thickness appear to have more influence in the capture and absorption of light than does epidermal cell shape.

Grapevine species from varied native habitats exhibit differences in embolism formation/repair associated with leaf gas exchange and root pressure.

Drought induces xylem embolism formation, but grapevines can refill non-functional vessels to restore transport capacity. It is unknown whether vulnerability to embolism formation and ability to repair differ among grapevine species. We analysed in vivo embolism formation and repair using x-ray computed microtomography in three wild grapevine species from varied native habitats (Vitis riparia, V. arizonica, V. champinii) and related responses to measurements of leaf gas exchange and root pressure. Vulnerability to embolism formation was greatest in V. riparia, intermediate in V. arizonica and lowest in V. champinii. After re-watering, embolism repair was rapid and pronounced in V. riparia and V. arizonica, but limited or negligible in V. champinii even after numerous days. Similarly, root pressure measured after re-watering was positively correlated with drought stress severity for V. riparia and V. arizonica (species exhibiting embolism repair) but not for V. champinii. Drought-induced reductions in transpiration were greatest for V. riparia and least in V. champinii. Recovery of transpiration after re-watering was delayed for all species, but was greatest for V. champinii and most rapid in V. arizonica. These species exhibit varied responses to drought stress that involve maintenance/recovery of xylem transport capacity coordinated with root pressure and gas exchange responses.

X-ray micro-tomography at the Advanced Light Source

The X-ray micro-Tomography Facility at the Advanced Light Source has been in operation since 2004. The source is a superconducting bend magnet of critical energy 11.5 keV; photon energy coverage is 8-45 KeV in monochromatic mode, and a filtered white light option yields useful photons up to 50 keV. A user-friendly graphical user interface allows users to collect tomographic and radiographic data sets with options including tiled and time series data sets. We will focus on recent projects that utilize sample environments for in-situ imaging. These environments include a high pressure triaxial flow cell which has allowed study of supercritical CO2 transport through brine-saturated sandstone at pressures typical of in-situ conditions for subsurface CO2 sequestration and water transportation within live plants.