Plant Vascular System

Phloem, which transport sugar produced in the leaves by photosynthesis to the rest of the plant, and xylem, which transport water and solutes from the roots to the leaves, represent the major components of the vasculature of a plant. Phloem and xylem are generally found together in vascular bundles, but can lie in various positions relative to each other.

Water in the xylem is maintained in a continuous thin column extending the entire height of the plant by intermolecular attractions whereby providing support. As water evaporates through stomata in the leaves, more water is drawn up through the roots, enabling a continual flow of water. The balancing of water intake and loss is vital to the health of the plant.

There has consequently been much study of the transport of water through the xylem. Not only has this provided interesting insights into the evolution of plants, but it has helped farmers and horticulturists improve the growth and hardiness of their plants, whereby increasing productivity¹.

Studying water transport in plants

One challenge to studying plant vascular function is that measurements can disturb the behaviour of the vascular system so that it does not reflect the system in its natural state, leading to artefacts. Although staining methods have provided some important insights into the patterns of water flow in plants, the technique is prone to artefacts. For example, the introduced dye may leach from one conduit to another giving rise to contact staining, which can hinder the study of fine scale patterns in some plant species.

In vivo imaging methods are thus being increasingly used to avoid or minimize the possibility of introducing artefacts. Magnetic resonance imaging (MRI) and high-resolution computed tomography (HRCT) are the imaging techniques most widely employed for visualising plant hydraulic function^2,3.

They provide a valuable tool for the study of plant vascular tissues as they readily distinguish fluid-filled conduits from gas-filled regions. HRCT, in particular, shows a strong distinction as fluid absorbs significantly more x-rays than gas. In addition, HRCT is becoming an increasingly popular choice of imaging tool for the study of water transport in plants due to the capacity to provide a greater level of detail, such as distinguishing cell walls from conduit lumens.

However, it has been shown that not all fluid-filled vessels are conductive^4,5. This may be because they are immature and not yet functional or because there is an obstruction, such as a gel or gum, preventing the flow of water. Consequently, current in vivo imaging may not allow for the simple identification of those conduits that are actively conducting water. Such knowledge is important when studying the flow of sap and comparing imaging results with other data that do not include non-functional vessels, such as direct hydraulic measurements.

The ability to identify conductive conduits from non-conductive conduits using in vivo imaging methods is thus highly desirable for plant research projects.

In vivo identification of conductive conduits

The differentiation between transporting and non-transporting plant vessels has been achieved using some in vivo nuclear MRI-based methods. These provided flow rates within the xylem and, also the phloem in some cases⁶. The utility of such systems however is limited since their resolution is not great enough to identify small individual conduits. Furthermore, commercial MRI equipment is not designed for the study of plants, and so it can be a challenge mounting the plant to be studied. It is usually necessary for the plant to be laid on its side so it cannot be studied in a natural state.

In addition, determination of conduits in an intact plant that are actively transporting water has been achieved using HRCT-based methods by adding a contrast agent that strongly absorbs X-rays. In this way, HRCT can be used in the evaluation of a wide range of tissues by enhancing contrast in regions where contrast would naturally be lacking.

A recent study demonstrated that addition of iohexol, a water-soluble iodine-rich molecule, as a contrast agent enabled in vivo identification of conductive conduits in intact plants using HRCT⁷. The plants selected for evaluation of the new technique were grapevine, for which non-functional vessels can appear to be functional in HRCT images, and American chestnut, which possesses a broad range of conduit sizes.

Having fed iohexol into the xylem stream, the plants were scanned using a Bruker Skyscan 2211 HRCT system to visualize flow paths within the xylem tissue and to identify conductive tracheary elements. In addition, the xylem of segments and gas-dried samples were stained to provide a comparison between intact scans and excised segments.

Iohexol was successfully transported through the xylem and marked conductive vessels on HRCT scans. The results obtained in vivo using iohexol were generally comparable to those obtained in stained cut segments, although iohexol allowed identification of a greater number of smaller conduits in some samples⁷.

It was possible to distinguish between gas-filled conduits, conductive conduits (containing iohexol tracer) and non-conductive conduits (no iohexol tracer present). The differentiation of conduit type showed only minimal differences between the visualisation in intact plants and the evaluation of excised segments.

Most importantly, both vessels and vasicentric tracheids were filled with iohexol in the chestnut plant, providing a new tool to study the functions of these different cell types⁷.

This latest research suggests that iohexol represents an important tracer for enabling the identification of conductive vessels in intact plants and may greatly improve the utility of HRCT as a tool in the study of plant hydraulic function.

References

1. Pratt RB, et al. Mortality of resprouting chaparral shrubs after a fire and during a record drought: physiological mechanisms and demographic consequences. Glob Chang Biol 2014;20:893-907.

2. Windt CW, et al. MRI of long-distance water transport: a comparison of the phloem and xylem flow characteristics and dynamics in poplar, castor bean, tomato and tobacco. Plant Cell Environ 2006;29:1715‑29.

3. Brodersen CR. Visualizing wood anatomy in three dimensions with high-resolution X-ray micro-tomography (μCT)-a review. IAWA J 2013;34:408‑424.

4. Hacke UG, et al. The standard centrifuge method accurately measures vulnerability curves of long-vesselled olive stems. New Phytol 2015;205:116‑127.

5. Jacobsen AL, et al. Functional lifespans of xylem vessels: development, hydraulic function, and post-function of vessels in several species of woody plants. Am J Bot 2018; doi:10.1002/ajb2.1029.

6. Hochberg U, et al. Stomatal closure, basal leaf embolism and shedding protect the hydraulic integrity of grape stems. Plant Physiol 2017;174:764‑775.

7. Pratt RB, Jacobsen AL. Identifying which conduits are moving water in woody plants: a new HRCT-based method. Tree Physiol. 2018 Apr 5. doi: 10.1093/treephys/tpy034. [Epub ahead of print].