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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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 physical dimensions of the HRCT instrument hutch at the Advanced Light Source limits live plants to ~1 m in height. As a consequence, imaging of live pl....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
See specifics listed above regarding equipment at the Advanced Light Source beamline 8.3.2