Trace Elemental Distribution Analysis in Plant Specimens using Laboratory Micro-XRF

Developing an understanding of how inorganic components are distributed throughout plants is of high importance to developing safe, more nutrient-rich food sources. An accurate understanding of plant nutrient uptake aids environmental and agricultural researchers with new insights in metallomics, including:

  • New phytoremediation techniques, in which plants are designed to remove toxic contaminants and recover the expanding amount of polluted land;
  • Agricultural studies, in which plant uptake of metals is modified to improve crop growth, increase the micronutrient value of the crop and reduce the absorption of toxic elements; and
  • Phytomining, in which “hyperaccumulating” plants can harvest precious minerals in an economic and environmentally-friendly way

While plant-based trace metal analysis is of high importance, it is difficult to carry out and often requires a synchrotron-based x-ray source. Synchrotrons produce brilliant, tunable x-ray beams, which facilitate sensitive, high-resolution microXRF; however, synchrotron beamlines are oversubscribed, and access generally requires a peer-reviewed application process and, if granted, travel time and expenses.

The AttoMap is a novel lab-based microXRF system designed by Sigray. This article covers the use of the AttoMap to research the uptake and partitioning of iron (Fe) in agricultural crops. Iron is one of the hardest micronutrients to analyze as it is present in very low (10-12 picogram) concentrations, meaning accuracy at a ppm level is required.

Iron plays a crucial role in the development of plants, being a key component in the reactions underpinning respiration and photosynthesis. Unfortunately, approximately 30% of the planet’s arable land is considered as iron-poor for crop growth.2

The following research found that OPT3 (Oligopetide Transporter 3), a transporter protein in plants, is used to regulate the loading of iron into developing leaves. This suggested that OPT3 proteins play a role in signaling iron demand between the plant shoots and roots.

AttoMap Micro-XRF mapping of a hyperaccumulating seedling. Larger view is a tricolor composite of K (red), Ni (blue), and Cl (green). Zoom-in of roots shows trace uptake of Mn (green) . Courtesy of Dr. Antony van der Ent and Dr. Peter Erskine, University of Queensland, Australia.

Figure 1. AttoMap Micro-XRF mapping of a hyperaccumulating seedling. Larger view is a tricolor composite of K (red), Ni (blue), and Cl (green). Zoom-in of roots shows trace uptake of Mn (green) . (Courtesy of Dr. Antony van der Ent and Dr. Peter Erskine, University of Queensland, Australia).

AttoMap Micro-XRF provides elemental imaging for multiple elements simultaneously. Left: tri-color composite of Zn (blue), Fe (green), and Ce (red). Right: individual channels for elements of interest. Courtesy of Cerege, CNRS, Aix-Marseille University.

Figure 2. AttoMap Micro-XRF provides elemental imaging for multiple elements simultaneously. Left: tri-color composite of Zn (blue), Fe (green), and Ce (red). Right: individual channels for elements of interest. (Courtesy of Cerege, CNRS, Aix-Marseille University).

Method

The research focused on the analysis of a genetically modified arabidopsis knockout (OPT3-3), provided by Prof. Olena Vatamaniuk (Associate Professor of Soil and Crop Sciences, Cornell University), alongside a wild type control with the aim of developing an understanding of OPT-3’s role.

Sample leaves were taken from the plants at different stages of their growth, with one leaf removed from the same plant following 16 growth days and another following 19 growth days. The Attomap microXRF was used to simultaneously analyze all of the elements present in order to determine how key minerals that impact plant growth (Ca, Mg, Fe, Zn and K) were distributed in the leaves.

For the leaf specimen taken after 16 days, mapping took place over an area of 3.5 mm x 3.8 mm with spot and step sizes of 10 μm (Figure 3). A tungsten (W) target, operating at 35 kV, was used in the x-ray’s multitarget source. It should be noted that tungsten was used in order to expand the analysis to a broad range of different elements; if only Fe (6.4 keV) was of interest, a copper (Cu) target would have more appropriate.

Further studies with an improved Fe sensitivity can be carried out with the AttoMap thanks to its unique multi-target x-ray source.

The mapping area for the leaf specimen taken after 19 days had an area of 4.0 mm x 8.3 mm with a 15 μm step size and a 10 μm step size (Figure 4). The same source settings as the 16-day leaf were used.

Left: tri-color composite of an opt3-3 mutant 16-day leaf: Fe (red), Ca (green), K (blue). Right: single-channel distribution maps of selected elements of interest. (Samples courtesy of Dr. Olena Vatamaniuk, Cornell University)

Figure 3. Left: tri-color composite of an opt3-3 mutant 16-day leaf: Fe (red), Ca (green), K (blue). Right: single-channel distribution maps of selected elements of interest. (Specimens courtesy of Dr. Olena Vatamaniuk, Cornell University)

Micro-XRF imaging of an opt3-3 mutant 19-day leaf. Tricolor composite on left shows: Fe (red), Ca (green), and K (blue). Single channel heat maps for a few selected elements of interest are shown on the right. (Samples courtesy of Dr. Olena Vatamaniuk, Cornell)

Figure 4. Micro-XRF imaging of an opt3-3 mutant 19-day leaf. Tricolor composite on left shows: Fe (red), Ca (green), and K (blue). Single channel heat maps for a few selected elements of interest are shown on the right. (Specimens courtesy of Dr. Olena Vatamaniuk, Cornell)

Results and Discussion

The experimental results showed that there were anomalies in trace-Fe distribution at the picogram-level in the knockout opt3-3 plant. In both the 16-day and 19-day leaf specimens it was found that the iron accumulated in the leaf’s minor veins, near the leaf blade periphery, and the pores (hydathodes), with the highest concentration in the central minor veins of the older leaf.

As the accumulation of Fe was occurring in locations where OPT3 is expressed at higher levels, the results suggest that OPT3 may play a key role in loading Fe back into the phloem, a type of vascular tissue that distributes nutrients and sugars from the leaves back towards the stems to assist in plant growth.

In contrast, the control leaf specimens had a markedly lower Fe distribution, with accumulation only observed at a small region on the furthest edge of the leaf.

Research carried out by Prof. Olena Vatamaniuk on the other elements involved in solute and water transportation in leaves (e.g. Ca and K) showed no statistical difference between the experimental (opt3-3) and control (wild type) samples. This suggests that other nutrients are not impacted by the presence of OPT3, supporting the hypothesis that OPT3 works specifically on Fe distribution pathways.

Summary

This research shows that new breakthroughs in lab-based microXRF instrumentation is allowing plant-based elemental analysis at a ppm level to take place without the need for a synchrotron.

In this research, the Sigray AttoMap was used to take picogram-scale measurements at a resolution smaller than the size of a cell (<10 μm). It was found that the AttoMap appeared to provide a better sensitivity towards K (3.3 keV) and Ca (3.7 keV) when compared to results previously collected using a synchrotron.

This is most likely the result of the AttoMap’s polychromatic beam, which can deliver better cross-sections than the 11 keV monochromatic beam used for the synchrotron experiments, confirming previous research that stated that “white light” beams are better for environmental analysis.3

Quantifying exactly to what extent the AttoMap improves the signal intensity for elements of a lower atomic number will be a subject of further research.

Not only does AttoMap allow the distribution of elements to be observed, but also the relative concentrations of each element that is detected. Additional exciting research that could be carried out using the AttoMap includes in vivo research where the elemental distribution in growing, living plants can be assessed.

This research is possible because the AttoMap uses a wide working distance (the distance between the sample and the source), which allows for uneven surfaces, such as leaves, or roots in soil to be imaged.

References

  1. HH Chu, et al. “Successful reproduction requires the function of Arabidopsis YELLOW STRIPE-LIKE1 and YELLOW STRIPE-LIKE3 metal-nicoti- anamine transporters in both vegetative and reproductive structures.” Plant Physiology 154 (2010): 197-210.
  2. Z Zhai, et al. “OPT3 is a Phloem-specific iron transporter that is essential for systemic iron signaling and redistribution of iron and cadmium in arabidopsis.” The Plant Cell 26 (2014): 2249-2264.
  3. SR Barberie, et al. “Evaluation of different synchrotron beamline configurations for x-ray fluorescence analysis of environmental samples.” Analytical Chemistry 86:16 (2014): 8253-8260.

This information has been sourced, reviewed and adapted from materials provided by Sigray, Inc.

For more information on this source, please visit Sigray, Inc.

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