Monitoring the Uptake and Localization of Organic Compounds in Plant Tissues Using a Hydroponic 14C-radiolabelling Assay and Phosphor Imaging

Summary

This article outlines a method for studying plant-mediated remediation using ¹⁴C-labelled organic contaminants. It describes hydroponic plant preparation, radiolabel uptake, and phosphor-imaging to track contaminant absorption, translocation, and metabolism. The technique offers a valuable tool for evaluating phytoremediation potential across various plant species and environmental pollutants.

Abstract

Phytoremediation, the use of plants to mitigate environmental contaminants, offers a sustainable and cost-effective approach to cleaning contaminated sites. Developing methods that aid in elucidating the mechanisms behind plant uptake and metabolism of pollutants is crucial for improving phytoremediation practices. This article describes a method to assess the uptake and transformation of organic contaminants by plants using radiolabeled compounds. ¹⁴C-labelled organic compounds, such as model ¹⁴C-naphthenic acids, are used to trace their absorption, translocation, localization, and metabolism in plant tissues. We have previously used this method with multiple plant species, including Elymus trachycaulus and Salix interior. These observations are corroborated here with the model plant, Arabidopsis thaliana, grown hydroponically in modified Hoagland solutions. Radiolabel uptake was monitored via liquid scintillation counting and phosphor-imaging, which allows for visualization and quantification of radiolabeled compounds within plant tissues. This method details the preparation of plant materials, the use of radiolabeled compounds, and the process of analyzing the distribution and fate of contaminants within plants. The method also includes strategies for assessing compound exudation and allows for the evaluation of both plant uptake and translocation of environmental contaminants. This approach provides insight into plant-mediated remediation processes and can be applied to the study of a wide range of environmental contaminants and plant species.

Introduction

Bioremediation is a remediation technology mediated by biological entities, such as plants and microorganisms. Phytoremediation, which uses plants or plant products to remove contaminants from the environment, can complement or serve as an alternative to microbial-based remediation, overcoming some of the limitations of using microorganisms alone^1,2,3,4. Phytoremediation is cost-effective due to the solar-driven processes of plants, where contaminants are absorbed through their roots or shoots and transported between cells and tissues where ....

Protocol

NOTE: Before proceeding with this protocol, it is important to consult the relevant institutional and national radioisotope safety guidelines and regulations. In brief, handle all radioactive material, or anything that may have come into contact with radioisotopes, while wearing double gloves, a lab coat, and safety goggles. The procedure was performed in a dedicated space in the lab with tape to mark off sections that contain radioisotopes. Also, after all procedures, clean the glassware and instruments immediately, then swab and test them for the presence of contaminating radioisotopes via scintillation counts. All materials and liquids used in these assays....

Results

The ¹⁴C-label uptake procedure is applicable to a variety of plant species beyond Arabidopsis, including Salix interior, Elymus trachycaulus (Link) Gould ex Shinners, and wetland species such as Juncus balticus^10,12. However, for representative purposes, we present results from Arabidopsis ecotype Columbia-7 (Col-7). Previously, we demonstrated that environmental contaminants, such as naphthen.......

Discussion

Phytoremediation has gained attention in recent decades as a passive, economical, and environmentally friendly method for cleaning industrial waste and contaminated sites¹. Identifying plant species with enhanced contaminant uptake and understanding the molecular processes governing organic molecule absorption could improve phytoremediation strategies. Unlike other indirect methods, the radiolabel uptake and phosphor imaging technique described here can be used widely to track the uptake and trans.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Hoagland Modified Basal Salt Mixture	Phyto Technology Laboratories	H353	Contains Ferrous Sulfate
Agar	BioShop	AGR001
MES (2-(N-morpholino) ethane sulfonic acid)	BioShop	MES555
pH Meter	Mettler Toledo	FiveEasy
Methanol	BioShop	MET302
12C-Adamantane carboxylic acid	Sigma Aldrich	106399
14C-Adamantane carboxylic acid	ViTrax		Custom synthesis
125 mL Erlenmeyer flasks	VWR	30623-174
50 mL Falcon tubes	VWR	CA21008-940
Microcentifuge tubes	VWR	87003-294
Plant Growth Chambers	Conviron
Tip Boxes	DiaMed	DIATEC530-8376
Rubbermaid Roughneck 3 Gal Tote	Amazon	B0B3345MYH
Filter paper	VWR	CA28320-020
Petri dish	VWR	11019-566
Shaker with flask holders	Forma Scientific	Model 4516
Scintillation cocktail	Research Products International	111195
Scintillation vials	VWR	10014-296
Scintillation counter	Beckman Coulter	LS 6000IC	vials used will depend on your scintillation counter
Gel dryer	Bio-Rad	Model 583
Laser scanning phosphor imager	Bio-Rad	Molecular Imager FX