A Method to Preserve Wetland Roots and Rhizospheres for Elemental Imaging

Summary

We describe a protocol to sample, preserve, and section intact roots and the surrounding rhizosphere soil from wetland environments using rice (Oryza sativa L.) as a model species. Once preserved, the sample can be analyzed using elemental imaging techniques, such as synchrotron X-ray fluorescence (XRF) chemical speciation imaging.

Abstract

Roots extensively interact with their soil environment but visualizing such interactions between roots and the surrounding rhizosphere is challenging. The rhizosphere chemistry of wetland plants is particularly challenging to capture because of steep oxygen gradients from the roots to the bulk soil. Here a protocol is described that effectively preserves root structure and rhizosphere chemistry of wetland plants through slam-freezing and freeze drying. Slam-freezing, where the sample is frozen between copper blocks pre-cooled with liquid nitrogen, minimizes root damage and sample distortion that can occur with flash-freezing while still minimizing chemical speciation changes. While sample distortion is still possible, the ability to obtain multiple samples quickly and with minimal cost increases the potential to obtain satisfactory samples and optimizes imaging time. The data show that this method is successful in preserving reduced arsenic species in rice roots and rhizospheres associated with iron plaques. This method can be adopted for studies of plant-soil relationships in a wide variety of wetland environments that span concentration ranges from trace-element cycling to phytoremediation applications.

Introduction

Roots and their rhizospheres are dynamic, heterogeneous, and critically important for understanding how plants obtain mineral nutrients and contaminants^1,2,3. Roots are the primary pathway by which nutrients (e.g., phosphorus) and contaminants (e.g., arsenic) move from soil to plants and thus understanding this process has implications for food quantity and quality, ecosystem functioning, and phytoremediation. However, roots are dynamic in space and time growing in response to nutrient acquisition needs and they often vary in function, diameter, and structure (e.g., lateral roots, adventitious roots, root hairs)². Heterogeneity of root systems can be studied on spatial scales from cellular to ecosystem-level and on temporal scales from hourly to decadal. Thus, the dynamic and heterogeneous nature of roots and their surrounding soil, or rhizosphere, poses challenges for capturing rhizosphere chemistry over time. Despite this challenge, it is imperative to study roots in their soil environment to characterize this critical plant-soil relationship.

The rhizosphere chemistry of wetland plants is particularly challenging to investigate because of steep oxygen gradients that exist from bulk soil to the roots, which change in space and time. Because roots need oxygen to respire, wetland plants have adapted to the low oxygen conditions of wetland soils by creating aerenchyma^{4, 5}. Aerenchyma are hollowed cortical tissues that extend from shoots to roots, allowing the diffusion of air through the plant into the roots. However, some of this air leaks into the rhizosphere in less suberized parts of the roots particularly near lateral root junctions, less mature root tips and elongation zones^6,7,8,9. This radial oxygen loss creates an oxidized zone in the rhizosphere of wetland plants that affects rhizosphere (bio-geo)chemistry and is distinct from the reduced bulk soil^10,11,12. To understand the fate and transport of nutrients and contaminants in wetland rhizospheres and roots, it is critical to preserve the chemically reduced bulk soil, the oxidized rhizosphere, and roots of wetland plants for analysis. However, because the bulk soil contains reduced soil constituents that are oxygen-sensitive, root and soil preservation methods must preserve root structures and minimize oxygen-sensitive reactions.

Methods exist to fix plant tissues and preserve the ultrastructure for imaging, but those methods cannot be applied to chemically preserve roots growing in wetland soil. For investigations where only the elemental distribution within plant cells is desired, plants are typically grown hydroponically and roots can be easily removed from solution, fixed under high-pressure freezing and freeze substitution and sectioned for a variety of imaging applications including high-resolution secondary ion mass spectrometry (nanoSIMS), electron microscopy, and synchrotron X-ray fluorescence (S-XRF) analysis^13,14,15. To investigate Fe plaque on the outside of wetland roots, these hydroponic studies must artificially induce Fe plaque formation in solution¹⁶, which does not accurately represent the heterogeneity of the distribution and mineral composition of Fe plaque formation and associated elements in situ^17,18,19,20. Methods exist to preserve wetland soil and associated microorganisms with freeze-coring²¹, but it is difficult to obtain roots with this technique. Current methods to visualize roots growing in soil and their rhizospheric chemistry consist of two primary measurement types: elemental fluxes and total elemental concentration (and speciation). The former is typically measured using diffusive gradients in thin films (DGT)^22,23,24, in which soil is placed into rhizoboxes to support plant growth in a laboratory setting and labile elements in the soil diffuse through a gel into a binding layer. This binding layer can then be imaged to quantify the labile elements of interest. This technique can successfully illustrate relationships between roots and the rhizosphere^24,25,26,27, but artefacts from root-bounding may exist by growing plants in rhizoboxes, and information on the root interior is not captured with DGT. The latter involves sampling of the roots and rhizosphere, preserving the sample, and directly analyzing elemental distribution on a sample section. For this environmental sampling of wetland plant roots and their surrounding rhizosphere, careful sample handling is required to avoid artefacts from sample preparation.

Here a protocol is described that effectively preserves root structures and rhizosphere chemistry of wetland plants by slam-freezing and freeze drying. Flash-freezing can drastically slow down transformations of oxygen sensitive solutes but may damage roots and may cause mobilization when samples dry out. However, slam-freezing where the sample is frozen between copper blocks pre-cooled with liquid nitrogen minimizes root damage and sample distortion²⁸. The preserved samples are then embedded in an epoxy resin that preserves As speciation^{20, 29} and can be cut and polished for imaging of roots within their rhizosphere soil. The samples in this report were analyzed by S-XRF chemical speciation imaging after thin sectioning. However, other imaging techniques could also be used, including laser ablation-inductively coupled plasma mass spectrometry (LA-ICP-MS), particle induced x-ray emission (PIXE), secondary ion mass spectrometry (SIMS), and laser induced breakdown spectroscopy (LIBS) imaging.

Protocol

1. Preparation of slam-freezing equipment

1. Place two copper blocks (~5 cm x 5 cm x 15 cm) horizontally inside of a clean cooler capable of holding liquid nitrogen and pour enough liquid nitrogen to submerge the blocks. Once the bubbling subsides, place two spacers on top of one copper block at each end.
   NOTE: The spacer height determines the height of the sample to be frozen; this example uses a 2 cm spacer to create cubes approximately 3 cm x 3 cm x 2 cm. The volume of the liquid nitrogen will depend on the cooler size. This example uses approximately 1 L for approximately 5 cubes in series.
   CAUTION: Use proper personal protective equipment and ventilation as liquid nitrogen is a cryogen and an asphyxiant.
2. Using tongs and cryogenic gloves, stand up the other copper block on its end, to make retrieval easier when the sample is in place.

2. Sample collection and slam-freezing

1. Extract the desired plant and rhizosphere from the wet soil using a shovel and ensure that the dug hole is much larger than the desired root volume. Place the soil and plant into a container and place it on a benchtop.
   NOTE: The entire potted soil and plant from a pot study can also be used.
2. Determine the desired soil location where roots are to be taken (i.e., depth and proximity to the shoot). Cut away excess soil using a steel blade, taking care not to disturb soil in the desired area. When the desired area is reached, cut a root "cube" approximately 3 cm x 3 cm x 2 cm and immediately place the cube between the two spacers on the horizontal copper block. Using cryogenic gloves, pick up the vertical copper block and place it on top of the spacers to slam-freeze the rhizosphere cube.
3. After bubbling subsides (~5 min), retrieve the slam-frozen rhizosphere cube from the copper blocks and wrap inside of a pre-labeled aluminum foil square. Mark the orientation of the block on the foil if desired. Place in a second container of liquid nitrogen until storage in a -80 °C freezer.
4. Repeat as needed to obtain the desired number of root cubes from the field site or the experiment. Ensure both copper blocks are given time to cool between samples.

3. Freeze-drying and embedding rhizosphere cubes

1. Prepare the freeze dryer according to the manufacturer's instructions. Take care to ensure it has obtained the proper vacuum pressure and temperature prior to removing samples from the -80 °C freezer.
2. When freeze dryer is ready to receive samples, place one frozen rhizosphere cube inside of a clean and acid-washed 50 mL tube and cover loosely with a clean disposable wipe. Secure the wipe with a rubber band. Repeat as needed to ensure one cube per tube.
   NOTE: If the sample is too large for a tube, it can be placed directly into the freeze dryer vessel using the aluminum foil as a sample holder.
3. Place tubes containing samples in freeze dryer vessels and freeze dry for several days. The exact drying time will depend on soil properties.
   NOTE: Store dried samples in the freeze dryer or a desiccator to avoid rehydration.
4. Use a steel blade to cut dried soil cubes to size so that they fit into the desired form (e.g., 25 mm diameter form is ideal for most applications). Label each form, place the soil cubes in the forms and place the forms inside a vacuum desiccator.
5. Prepare epoxy according to manufacturer's instructions. Ensure that the chosen epoxy is not contaminated with and does not cause speciation changes of desired elements ^{20, 29, 30}.
6. Use a dropper to add epoxy to the form on one side of the soil, till it entirely covers the sample. The soil will darken in color as the epoxy wets the soil.
   NOTE: Add the epoxy slowly to allow the air in the soil to escape.
7. Once forms are filled with epoxy, close the vacuum desiccator and turn on the vacuum. Depending on the amount of air trapped in the soil, more epoxy may need to be added to the forms periodically. Check the level of epoxy every 30-90 min for the first 1-4 h and add epoxy as needed.
8. Remove the sample from the form once the epoxy has hardened (~5 days).

4. Cutting and sectioning the rhizosphere cubes

1. Cut the sample using a diamond blade precision wet saw. Cut the samples in different locations if no roots are obtained in the previous cut.
2. Manually sand the cut samples with progressively finer sandpaper (e.g., 220, 500, 1000, and 1500 grit) on the cut side for ~30 s.
3. Perform surface imaging of the samples using techniques such as LA-ICP-MS.
   NOTE: To prepare thin sections for S-XRF, either send the samples out to a company capable of preparing the thin sections (single or double side polishing) or follow the steps 4.4 - 4.6 as described below.
4. Glue the desired sample side to a quartz slide using super glue and allow to cure overnight.
5. Using a thin sectioning machine, cut soil on slides to 2 mm thick and then grind to the desired thickness (typically 30 µm). The sample surface can be polished if desired.
6. Perform S-XRF imaging of the sections. Follow the appropriate steps at the desired synchrotron facility and beamline to apply for and utilize imaging time.

Results

This method allows for preservation of roots and chemical species in the roots and rhizosphere of wetland plants and into the bulk soil. In this work, the method was used to evaluate As speciation and co-localization with Fe and Mn oxides and plant nutrients in the rhizosphere of rice (Oryza sativa L.). Rice was grown at the RICE Facility at the University of Delaware where 30 rice paddy mesocosms (2 m x 2 m, 49 plants each) are used to grow rice under various soil and water management conditions with the goal o...

Discussion

This paper describes a protocol to obtain preserved bulk soil + rhizospheres of wetland plant roots using a slam-freezing technique that can be used for elemental imaging and/or chemical speciation mapping.

There are several benefits of this method over existing methods. First, this method allows the simultaneous investigation of roots and the surrounding rhizospheres. Methods currently exist to preserve and chemically image roots out of their soil environment by washing away the soil and pres...

Materials

Name	Company	Catalog Number	Comments
Copper blocks	McMaster Carr	89275K42
Diamond blade	Buehler	15 LC, 102 mm x 0.3 mm	operation speed: 225 rpm
Epoxy forms	Struers	40300085	FixiForm
Epoxy	Epotek	301-2FL
Superglue	Loctite	404
Thin sectioning machine	Buehler	PetroThin
Wet saw	Buehler	IsoMet 1000