JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

Published: April 5th, 2013

DOI:

10.3791/50162

1U.S. Department of Agriculture, 2Department of Viticulture and Enology, University of California - Davis, 3Hawkesbury Institute for the Environment, University of Western Sydney, 4Advanced Light Source, Lawrence Berkeley National Lab, 5Citrus Research & Education Center, University of Florida

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods.

Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

Water is transported from plant roots to the leaves in a vascular tissue called xylem - a network of interconnected conduits, fibers, and living, metabolically active cells. Transport function of plant xylem must be maintained to supply nutrients and water to leaves for photosynthesis, growth, and ultimately survival. Water transport in xylem conduits can be disrupted when the xylem network is compromised by pathogenic organisms. In response to such infections plants often produce gels, gums, and tyloses as a means to isolate pathogen spread (e.g. McElrone et al 2008; 2010). Drought stress can also limit water transport in xylem. As plants lose wate....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Protocol details described below were written specifically for work at the Advanced Light Source 8.3.2 beamline. Adaptations may be required for work at other synchrotron facilities. Proper safety and radiation training is required for use of these facilities.

1. Sample Preparation for Live Plants

  1. Grow plants in ~10 cm diameter pots, and ensure that the main stem (or portion of the plant to be scanned) is as centered as possible and oriented vertically in the pot. The phys.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Synchotron HRCT scans have been successfully implemented on a wide variety of plant tissues and species using beamline 8.3.2 (Figure 5), and have provided new insights into the structure and function of plant xylem at unprecedented resolution in 3D. The visualization and exploration capabilities provided by the 3D reconstructions (as illustrated in Figures 6-8; and Movies 1-3) allow for precise determination of location and orientation of structures with the xylem networks on both.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Synchotron HRCT provides plant biologists with a powerful, non-destructive tool to explore the inner workings of plant vasculature in incredible detail. This technology has been used recently to identify previously undescribed anatomical structures in grapevine xylem that differentially alter xylem network connectivity in various grapevine species (Brodersen et al. 2012b, in press)- this connectivity can drastically alter the ability of vascular pathogens and emboli to spread destructively throughout xyle.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

The authors would like to thank S Castorani, AJ Eustis, GA Gambetta, CM Manuck, Z Nasafi, and A Zedan. This work was funded by: the U.S. Department of Agriculture-Agricultural Research Service Current Research Information System funding (research project no. 5306-21220-004-00; The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.); and NIFA Specialty crops research initiative grant to AJM.

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
Material Name/Equipment Company Catalogue Number Comments (optional)
See specifics listed above regarding equipment at the Advanced Light Source beamline 8.3.2

  1. Brodersen, C. R., McElrone, A. J., Choat, B., Matthews, M. A., Shackel, K. A. The dynamics of embolism repair in xylem: in vivo visualizations using high resolution computed tomography. Plant Physiology. 154, 1088-1095 (2010).
  2. Brodersen, C. R., Lee, E., Choat, B., Jansen, S., Phillips, R. J., Shackel, K. A., McElrone, A. J., Matthews, M. A. Automated analysis of 3D xylem networks using high resolution computed tomography (HRCT). New Phytologist. 191 (4), 1168-1179 (2011).
  3. Brodersen, C., Roark, L., Pittermann, J. The physiological implications of primary xylem organization in two ferns. Plant, Cell & Environment. , (2012).
  4. Brodersen, C., Choat, B., Chatelet, D., Shackel, K. A., Matthews, M. A., McElrone, A. J. Conductive xylem bridges contribute differentially to radial connectivity in grapevine stems (Vitis vinifera and V. arizonica). American Journal of Botany. , (2012).
  5. McElrone, A. J., Jackson, S., Habdas, P. Hydraulic disruption and passive migration by a bacterial pathogen in oak tree xylem. Journal of Experimental Botany. 59, 2649-2657 (2008).
  6. McElrone, A. J., Grant, J., Kluepfel, D. The role of ethylene-induced tyloses in canopy hydraulic failure of mature walnut trees afflicted with apoplexy disorder. Tree Physiology. 30, 761-772 (2010).
  7. Tyree, M., Sperry, J. Vulnerability of xylem to cavitation and embolism. Annual Review of Plant Biology. 40 (1), 19-36 (1989).
  8. Tyree, M., Zimmermann, M. . Xylem structure and the ascent of sap. , (2002).
  9. Holbrook, N. M., Zwienieck, M. A. . Vascular Transport in Plants. , (2005).
  10. Mayo, S. C., Chen, F., Evans, F. Micron-scale 3D imaging of wood and plant microstructure using high-resolution x-ray phase-contrast microtomography. Journal of Structural Biology. 171, 182-188 (2010).
  11. Mannes, D., Marone, F., et al. Application areas of synchrotron radiation tomographic microscopy for wood research. Wood Science and Technology. 44, 67-84 (2010).
  12. Maeda, E., Miyake, H. A non-destructive tracing with an x-ray micro ct scanner of vascular bundles in the ear axes at the base of the lower level rachis-branches in japonica type rice (oryza sativa. Japanese Journal of Crop Science. 78 (3), 382-386 (2009).
  13. Steppe, K., Cnudde, V., et al. Use of x-ray computed microtomography for non-invasive determination of wood anatomical characteristics. Journal of Structural Biology. 148 (1), 11-21 (2004).
  14. Zimmermann, M. Dicotyledonous wood structure (made apparent by sequential sections). Encyclopaedia Cinematographica. , (1971).
  15. Lee, E. F., Brodersen, C. R., McElrone, A. J., et al. Analysis of HRCT-derived xylem network reveals reverse flow in some vessels. , (2013).
  16. McDowell, N. G., Pockman, W. T., et al. Mechanisms of plant survival and mortality during drought: why do some plants survive while others succumb. New Phytologist. 178, 719-739 (2008).
  17. McElrone, A. J., Brodersen, C. R., et al. Centrifuge technique consistently overestimates vulnerability to water-stress induced cavitation in grapevines as confirmed with high resolution computed tomography. New Phytologist. , (2012).
  18. Lee, K., Avondo, J., et al. Visualizing plant development and gene expression in three dimensions using optical projection tomography. Plant Cell. 18, 2145-2156 (2006).
  19. Truernit, E., Bauby, H., et al. High-resolution whole-mount imaging of three-dimensional tissue organization and gene expression enables the study of phloem development and structure in Arabidopsis. Plant Cell. 20, 1494-1503 (2008).
  20. Jahnke, S., Menzel, M. I., et al. Combined MRI-PET dissects dynamic changes in plant structures and functions. The Plant Journal. 59, 634-644 (2009).
  21. Iyer-Pascuzzi, A. S., Symonova, O., et al. Imaging and analysis platform for automatic phenotyping and trait ranking of plant root systems. , (2010).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved