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Plants require many micronutrients and macronutrients to carry out their vital functions. Analysis of these elements helps understand their role in plant growth and development. Investigating these elements in plants helps scientists to assess the nutritional requirement, which could, in turn, be vital for the enhancement of crop productivity. Elemental analysis can also determine the presence of harmful contaminants such as heavy metals in plants. Early determination of such elements could significantly reduce the introduction of harmful contaminants in the food chain and protect the environment.
Elemental analysis has proved to be an important tool to answer many critical research questions, especially in agronomy, plant molecular biology, plant physiology, ionomics, and plant nutrition.
Importance of Elemental Analysis in Plant Research
Researchers believe that understanding the metallic distribution in plants would help improve plant mineral content and productivity. They have effectively characterized micronutrients and macronutrients and linked them with genes. For example, the distribution of metals such as iron and manganese have been studied in Arabidopsis seeds using X-ray fluorescence microscopy (XFM). Their tolerance to metals was also associated with the presence of the metal tolerance protein 8 (MTP8) gene.
The elemental analysis could also help to enhance human nutrition. The nutritional value of food depends not only on the total mineral concentration but also on its distribution and speciation within the tissues containing the food particles. For example, researchers have revealed that the inner layers of the pericarp of buckwheat grains are rich in potassium, manganese, calcium, and iron while the outer layer is rich in sodium, magnesium, phosphorus, and aluminum.
Scientists use various elemental analyses to cognize metals that are toxic to plants. This approach also helps understand the mechanism behind the plants’ metallic tolerance. For instance, researchers used NanoSIMS to unveil the distribution of aluminum in root tissues of Glycine max and understand how it exerts toxicity to the plant. Imaging techniques, such as XFM, are also used to determine the impact of hyperaccumulation of metals in plants.
Elemental Analysis Used in Plant Research
Several techniques are used to evaluate the distribution of elements within plants, which have their benefits and disadvantages. These techniques usually vary by the range of elements they can analyze, resolving power, quantitative analysis, and the sample preparation (fresh or freeze-dried). Some of the elemental analysis techniques used in plant research are discussed below:
X-Ray Fluorescence-Based Techniques
These techniques are based on the element’s characteristic reaction to fluorescent x-rays. When fluorescent x-rays such as high-energy x-rays (XFM), electrons (SEM- and TEM-EDS), or protons (PIXE) are introduced to a metallic sample through a focused beam it excites the elements. The degree by which an element gets excited depends on the energy of the incident x-rays, electrons, or protons, which is detected and quantified by a detector. Although in principle, an incident x-ray beam does not destroy the sample, the generation of the free radicals might have a damaging effect on the tissues being analyzed. However, contrary to electron and proton beams, x-ray beams do not produce heat in the sample being analyzed.
As the name suggests, the scanning emission microscopy- energy-dispersive X-ray spectroscopy (SEM-EDS) and transmission emission microscopy- energy dispersive X-ray spectroscopy (TEM-EDS) uses an electron beam to scan samples. This is the most commonly used method which determines the elemental distribution in plant tissues. A cryo-SEM system is used to study frozen plant tissue specimens in the hydrated state. However, in the study of the elemental composition of plant tissues, the SEM-EDS method is not accurate owing to the interaction of the electrons with the sample. In this regard, TEM-EDS is a better approach.
XFM (both synchrotrons or laboratory-based) generate fluorescent x-rays for elemental mapping. For plant analysis, the most popularly used XFM beamlines are Australian Synchrotron (Australia) and D21 at the European Synchrotron Radiation Facility (France). Researchers use XFM to study the elemental distribution throughout the thickness of the plant tissues.
This technique is most suitable for the analysis of elements such as manganese, iron, and zinc. Using the GeoPIXE software, the synchrotron-based XFM is used to quantify the elements present in the plant tissues. In micropixel, an ion beam of protons is used to produce fluorescent x-rays in the sample. PIXE is used in the imaging of metals (e.g., cadmium) and metalloids (e.g., antimony) which cannot be detected via synchrotron XFM.
Mass Spectrometry-Based Techniques
These techniques are highly sensitive, but as small portions of samples are removed for analysis, mass spectrometry-based methods are considered destructive. Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS) offers multi-element analyses with a wide range of metals (lithium to uranium) with modest resolution. This technique is highly beneficial for the analysis of both hydrated and dehydrated samples. LA-ICP-MS has been used for the imaging of iron, zinc, and manganese in the roots of Arabidopsis.
In NanoSIMS, ions are used as the incident beam, which collides with the sample surface causing atoms, ions, and molecules from the sample surface to sputter and, thereby, are ejected into the vacuum. This technique has been used to determine the mechanism by which foliar-applied zinc fertilizer translocate across the leaf surface.
Since 1920, autoradiography is being used in plant research. In this technique, radioactive isotopes are introduced to a plant, which gets redistributed throughout the plant tissues. These isotopes are easily traced and are studied by researchers. Autoradiography can detect one element (e.g., 55Fe, 59Ni, 32P, etc.) at a time and is mainly used for in vivo experiments.
Laser Confocal Microscopy
The laser confocal microscopy is based on the binding of ion-selective fluorophores to the targeted element. The detection is based on the excitation of the element using a specific wavelength emitted by lasers. This is also a non-destructive approach where the hydrated samples as well as in vivo analyses of living plants can be conducted. Recently, this method has been used for imaging the distribution of Ni2+ in Alyssum murale.
The study of elemental distribution in the plant tissues is extremely important as this helps determine the mineral and nutritional content of plants. Understanding the role of elements in crop development will ensure better productivity and plant growth.
References and Further Readings
Kopittke, P. M., et al. (2020). Methods to Visualize Elements in Plants. Plant physiology, 182(4), pp. 1869–1882. https://doi.org/10.1104/pp.19.01306
Borges, C.S. et al. (2020). Foliar Elemental Analysis of Brazilian Crops via Portable X-ray Fluorescence Spectrometry. Sensors, 20(9), p.2509. Available at: http://dx.doi.org/10.3390/s20092509
Haidu, D. et al. (2017). Elemental Characterization of Romanian Crop Medicinal Plants by Neutron Activation Analysis. Journal of Analytical Methods in Chemistry, Article ID 9748413. Available at: https://doi.org/10.1155/2017/9748413