Multi-element bioimaging of Arabidopsis thaliana roots

Abstract

(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...

Indications of Selenium Protection against Cadmium and Lead Toxicity in Oilseed Rape (Brassica napus L.)

“…Some crops convert Se mainly into SeMet and incorporate it into protein in place of methionine (Met) (Bañuelos et al, 2011; Persson et al, 2012, 2016a,b; Winkel et al, 2012; Yuan et al, 2013). In this study, the level of Se-Met was higher in the Cd and Pb treatments than in control, which suggests the protective role of Se against Cd and Pb toxicity might depend on the competition between Se and heavy metals for binding with the functional bioligands.…”

“…Furthermore, the presence of Cd and Pb in the growth medium has a great effect on Se speciation transformed by plants, which might be also considered as one of the important conditions of Se-induced Cd and Pb tolerance in oilseed rape plants. Further studies are necessary to determine that distribution and speciation of Se in oilseed rape plant cells using synchrotron-based techniques, such as X-ray fluorescence and X-ray absorption spectrometry, and mass spectrometry-based techniques, such as secondary ion mass spectrometry and laser-ablation inductively coupled plasma mass spectrometry (Zhao et al, 2014; Persson et al, 2016a,b). …”

Variation in the angiosperm ionome

“…The ionome of a particular plant, organ, tissue, cell or subcellular structure is affected by the phytoavailability of elements in the environment, transport processes into and within the plant, plant metabolism and the requirements of specific extracellular and intracellular structures (White and Brown , Baxter ). The ionome differs between plant species growing in the same environment (Broadley et al , White et al , Watanabe et al ), organs, tissues and cells of an individual plant (Sorreano , Cakmak et al , Conn and Gilliham , Subramanian et al , Baxter et al , Eggert and von Wirén , Zhao et al , He et al , Persson et al , Thomas et al , Watanabe et al , de la Fuente et al , White and Pongrac ) and extracellular and intracellular compartments (Sterner and Elser , Hawkesford et al , Broadley et al , , White et al ). Ultimately, differences in the ionomes of plants growing under identical conditions reflect differences in their genomes and gene expression that have evolved through mutation and the selection of adapted phenotypes.…”

Elevated root nicotianamine concentrations are critical for Zn hyperaccumulation across diverse edaphic environments

“…The observed effects on leaf Zn and Fe are consistent with data reported for A . thaliana (Klatte et al, ; Persson et al, ; Schuler, Rellán‐Álvarez, Fink‐Straube, Abadía, & Bauer, ). The effect on leaf Cu content is consistent with phenotypes of the NA‐free tomato mutant chloronerva (Pich & Scholz, ).…”

“…Moreover, the direct visualization of Zn across A . thaliana roots via laser ablation ICP‐MS revealed enhanced Zn accumulation around the vasculature of NA‐deficient plants and a concomitant decrease in the concentration of Zn in the xylem sap (Persson et al, ).…”