Multi-element bioimaging of Arabidopsis thaliana roots

Better understanding of root function is central for development of plants with more efficient nutrient uptake and translocation. We here present a method for multi-element bioimaging at the cellular level in roots of the genetic model system Arabidopsis thaliana. Using conventional protocols for microscopy we observed that diffusible ions such as potassium (K+) and sodium (Na+) were lost during sample dehydration. Thus, we developed a protocol which preserves ions in their native, cellular environment. Briefly, fresh roots are encapsulated in paraffin, then cryo-sectioned and freeze dried. Samples are finally analyzed by Laser Ablation-Inductively Coupled Plasma-Mass Spectrometry (LA-ICP-MS), utilizing a specially designed internal standard procedure. The method can be further developed to maintain the native composition of proteins, enzymes, RNA and DNA, making it attractive in combination with other omics techniques. To demonstrate the potential of the method we analyzed a mutant of A. thaliana unable to synthesize the metal chelator nicotianamine (NA). The mutant accumulated substantially more zinc (Zn) and manganese (Mn) than the wild type in the tissues surrounding the vascular cylinder. For iron (Fe) the images looked completely different, with Fe bound mainly in the epidermis of the WT plants, but confined to the cortical cell walls of the mutant. The method offers the power of ICP-MS to be fully employed, thereby providing a basis for detailed studies of ion transport in roots. Being applicable to A. thaliana, the molecular and genetic approaches available in this system can now be fully exploited in order to gain a better mechanistic understanding of these processes

Simultaneous iron, zinc, sulphur and phosphorus speciation analysis of barley grain tissues using SEC-ICP-MS and IP-ICP-MS

The increasing prevalence of iron (Fe) and zinc (Zn) deficiencies in human populations worldwide has stressed the need for more information about the distribution and chemical speciation of these elements in cereal products. In order to investigate these aspects, barley grains were fractionated into awns, embryo, bran and endosperm and analysed for Fe and Zn. Simultaneously, phosphorus (P) and sulfur (S) were determined since these elements are major constituents of phytic acid and proteins, respectively, compounds which are potentially involved in Fe and Zn binding. A novel analytical method was developed in which oxygen was added to the octopole reaction cell of the ICP-MS. This approach greatly improved the sensitivity of sulfur, measured as 48SO+. Simultaneously, Fe was measured as 72FeO+, P as 47PO+, and Zn as 66Zn+, enabling sensitive and simultaneous analysis of these four elements. The highest concentrations of Zn, Fe, S and P were found in the bran and embryo fractions. Further analysis of the embryo using SEC-ICP-MS revealed that the speciation of Fe and Zn differed. The majority of Fe co-eluted with P as a species with the apparent mass of 12.3 kDa, whereas the majority of Zn co-eluted with S as a 3 kDa species, devoid of any co-eluting P. Subsequent ion pairing chromatography of the Fe/P peak showed that phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate: IP6) was the main Fe binding ligand, with the stoichiometry Fe4(IP6)18. When incubating the embryo tissue with phytase, the enzyme responsible for degradation of phytic acid, the extraction efficiency of both Fe and P was doubled, whereas that of Zn and S was unaffected. Protein degradation on the other hand, using protease XIV, boosted the extraction of Zn and S, but not that of Fe and P. It is concluded that Fe and Zn have a different speciation in cereal grain tissues; Zn appears to be mainly bound to peptides, while Fe is mainly associated with phytic acid

Leaf Scorching following Foliar Fertilization of Wheat with Urea or Urea–Ammonium Nitrate Is Caused by Ammonium Toxicity

Foliar fertilization is a potential tool to increase the use-efficiency of nitrogen (N) fertilizers. However, whilst leaf scorching has frequently been reported, the underlying physiological processes are not clear. In the present work, we investigate the intensity of leaf scorching as affected by the balance between ammonium assimilation and accumulation. Leaves were sprayed with urea–ammonium nitrate (UAN) solution without surfactant or applied liquid droplets of urea in different N concentrations with surfactant. UAN solutions without surfactant containing >10% N caused leaf scorching already after 24 h and the severity increased with the N concentration. The same pattern was observed 3 days after the application of urea solutions containing >4% N together with surfactant. The scorching was accompanied by a massive increase in foliar and apoplastic ammonium (NH4+) concentration. Moreover, the activity of glutamine synthetase (GS), most pronouncedly that of the chloroplastic isoform (GS2), decreased a few hours after the application of high N-concentrations. Along with this, the concentration of glutamate—the substrate for GS—decreased. We conclude that leaf scorching is promoted by NH4+ accumulation due to a limitation in N assimilation capacity

The Intensity of Manganese Deficiency Strongly Affects Root Endodermal Suberization and Ion Homeostasis

Manganese (Mn) deficiency affects various processes in plant shoots. However, the functions of Mn in roots and the processesinvolved in root adaptation to Mn deficiency are largely unresolved. Here, we show that the suberization of endodermal cells inbarley (Hordeum vulgare) roots is altered in response to Mn deficiency, and that the intensity of Mn deficiency ultimatelydetermines whether suberization increases or decreases. Mild Mn deficiency increased the length of the unsuberized zoneclose to the root tip, and increased the distance from the root tip at which the fully suberized zone developed. By contrast,strong Mn deficiency increased suberization closer to the root tip. Upon Mn resupply, suberization was identical to that seen onMn-replete plants. Bioimaging and xylem sap analyses suggest that the reduced suberization in mildly Mn-deficient plantspromotes radial Mn transport across the endodermis at a greater distance from the root tip. Less suberin also favors the inwardsradial transport of calcium and sodium, but negatively affects the potassium concentration in the stele. During strong Mndeficiency, Mn uptake was directed toward the root tip. Enhanced suberization provides a mechanism to prevent absorbedMn from leaking out of the stele. With more suberin, the inward radial transport of calcium and sodium decreases, whereas thatof potassium increases. We conclude that changes in suberization in response to the intensity of Mn deficiency have a strongeffect on root ion homeostasis and ion translocation

Multi-elemental fingerprinting of plant tissue by semi-quantitative ICP-MS and chemometrics

The multi-elemental capacity of Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) is rarely fully utilized in traditional full-quantitative analysis. The main obstacles are limited availability of multi-elemental standards and the need for time-consuming external calibrations. In this study, a novel semi-quantitative quadrupole ICP-MS based method for multi-elemental fingerprinting of plant tissue was developed as a high-throughput alternative to full-quantitative analysis. The main analytical objectives were low data acquisition time (70%. In conjunction with chemometrics, the discrimination power of the semi-quantitative results was better than that of full-quantitative analysis. The superior discrimination power of semi-quantitative analysis was maintained, even when it was combined with a high-throughput digestion procedure, which represented a 5 fold reduction in analytical labour consumption. Thus, the large amount of elemental information obtained using semi-quantitative ICP-MS fully outweighed the lack of accuracy compared to full-quantitative analysis. For the first time it is demonstrated that semi-quantitative ICP-MS in combination with chemometrics provides a fast and powerful alternative to traditional full-quantitative ICP-MS. The method developed here constitutes a promising novel analytical tool, which has the potential to mature into a routine procedure for testing e.g. the authenticity and adulteration of food products