(J.K.S.); 0000-0003-2020-1902 (S.H.).Better understanding of root function is central for the development of plants with more efficient nutrient uptake and translocation. We here present a method for multielement bioimaging at the cellular level in roots of the genetic model system Arabidopsis (Arabidopsis thaliana). Using conventional protocols for microscopy, we observed that diffusible ions such as potassium and sodium were lost during sample dehydration. Thus, we developed a protocol that preserves ions in their native, cellular environment. Briefly, fresh roots are encapsulated in paraffin, cryo-sectioned, and freeze dried. Samples are finally analyzed by laser ablation-inductively coupled plasma-mass spectrometry, 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 Arabidopsis unable to synthesize the metal chelator nicotianamine. The mutant accumulated substantially more zinc and manganese than the wild type in the tissues surrounding the vascular cylinder. For iron, the images looked completely different, with iron bound mainly in the epidermis of the wild-type plants but confined to the cortical cell walls of the mutant. The method offers the power of inductively coupled plasma-mass spectrometry to be fully employed, thereby providing a basis for detailed studies of ion transport in roots. Being applicable to Arabidopsis, 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.Investigations of the localization of inorganic elements in young plant roots may answer a range of important and unresolved questions with respect to root functionality and plant nutrient transport. To date, our understanding of how plants control the radial root transport of essential plant nutrients and toxic elements is mainly circumstantial, relying on changes in shoot or shoot-to-root concentration ratios or analyses of xylem sap composition. Roots of Arabidopsis (Arabidopsis thaliana) have a simple cellular organization and are unrivaled in their ability to be imaged by confocal microscopy, as they are very thin (diameter approximately 120 mm) and have a low background fluorescence. This has led to an amazingly detailed understanding of the growth and development of roots. Unfortunately, the fragile nature of these roots constitutes a major challenge when trying to understand the processes that drive nutrient uptake at the same level of detail. The method we present here for element bioimaging of Arabidopsis roots is a critical step in utilizing the potential of combining targeted genetic modifications and bioimaging at the cellular level in order to unravel the complexities of how roots selectively acquire and translocate mineral nutrients from the soil.The uptake and radial tr...

Manganese (Mn) deficiency affects various processes in plant shoots. However, the functions of Mn in roots and the processes involved in root adaptation to Mn deficiency are largely unresolved. Here, we show that the suberization of endodermal cells in barley (Hordeum vulgare) roots is altered in response to Mn deficiency, and that the intensity of Mn deficiency ultimately determines whether suberization increases or decreases. Mild Mn deficiency increased the length of the unsuberized zone close 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 on Mn-replete plants. Bioimaging and xylem sap analyses suggest that the reduced suberization in mildly Mn-deficient plants promotes radial Mn transport across the endodermis at a greater distance from the root tip. Less suberin also favors the inwards radial transport of calcium and sodium, but negatively affects the potassium concentration in the stele. During strong Mn deficiency, Mn uptake was directed toward the root tip. Enhanced suberization provides a mechanism to prevent absorbed Mn from leaking out of the stele. With more suberin, the inward radial transport of calcium and sodium decreases, whereas that of potassium increases. We conclude that changes in suberization in response to the intensity of Mn deficiency have a strong effect on root ion homeostasis and ion translocation.

Background: To understand processes regulating nutrient homeostasis at the single-cell level there is a need for new methods that allow multi-element profiling of biological samples ultimately only available as isolated tissues or cells, typically in nanogram-sized samples. Apart from tissue isolation, the main challenges for such analyses are to obtain a complete and homogeneous digestion of each sample, to keep sample dilution at a minimum and to produce accurate and reproducible results. In particular, determining the weight of small samples becomes increasingly challenging when the sample amount decreases. Results: We developed a novel method for sampling, digestion and multi-element analysis of nanogram-sized plant tissue, along with strategies to quantify element concentrations in samples too small to be weighed. The method is based on tissue isolation by laser capture microdissection (LCM), followed by pressurized micro-digestion and ICP-MS analysis, the latter utilizing a stable µL min −1 sample aspiration system. The method allowed for isolation, digestion and analysis of micro-dissected tissues from barley roots with an estimated sample weight of only ~ 400 ng. In the collection and analysis steps, a number of contamination sources were identified. Following elimination of these sources, several elements, including magnesium (Mg), phosphorus (P), potassium (K) and manganese (Mn), could be quantified. By measuring the exact area and thickness of each of the micro-dissected tissues, their volume was calculated. Combined with an estimated sample density, the sample weights could subsequently be calculated and the fact that these samples were too small to be weighed could thereby be circumvented. The method was further documented by analysis of Arabidopsis seeds (~ 20 µg) as well as tissue fractions of such seeds (~ 10 µg). Conclusions: The presented method enables collection and multi-element analysis of small-sized biological samples, ranging down to the nanogram level. As such, the method paves the road for single cell and tissue-specific quantitative ionomics, which allow for future transcriptional, proteomic and metabolomic data to be correlated with ionomic profiles. Such analyses will deepen our understanding of how the elemental composition of plants is regulated, e.g. by transporter proteins and physical barriers (i.e. the Casparian strip and suberin lamellae in the root endodermis).