A rapid total reflection X-ray fluorescence protocol for micro analyses of ion profiles in Arabidopsis thaliana

The ion homeostasis of macro and micronutrients in plant cells and tissues is a fundamental requirement for vital biochemical pathways including photosynthesis. In nature, ion homeostasis is affected mainly by three processes: 1. Environmental stress factors, 2. Developmental effects, and 3. Loss or gain-of-function mutations in the plant genome. Here we present a rapid total reflection X-ray fluorescence (TXRF) protocol that allows for simultaneous quantification of several elements such as potassium (K), calcium (Ca), sulfur (S), manganese (Mn) and strontium (Sr) in Arabidopsis thaliana leaf specimens. Our procedure is cost-efficient and enables precise, robust and highly reproducible measurements on tissue samples as small as 0.3 mg dry weight. As shown here, we apply the TXRF procedure to detect accurately the early replacement of K by Na ions in leaves of plants exposed to soil salinity, a globally increasing abiotic stress factor. Furthermore, we were able to prove the existence of a leaf development-dependent ion gradient for K, Ca, and other divalent ions in A. thaliana; i.e. old leaves contain significantly lower K but higher Ca than young leaves. Lastly, we show that our procedure can be readily applied to reveal subtle differences in tissue-specific ion contents of plant mutants. We employed independent A. thaliana kea1kea2 loss-of-function mutants that lack KEA1 and KEA2, two highly active chloroplast K exchange proteins. We found significantly increased K levels specifically in kea1kea2 mutants, i.e. 55 mg ∗ g⁻¹ dry weight, compared to 40 mg ∗ g⁻¹ dry weight in wild type plants. The TXRF procedure can be supplemented with Flame atomic absorption (FAAS) and emission spectrometry (FAES) to expand the detection range to sodium (Na) and magnesium (Mg). Because of the small sample amounts required, this method is especially suited to probe individual leaves in single plants or even specific leaf areas. Therefore, TXRF represents a powerful method to gain detailed quantitative insights into I) the effect of environmental stress on plant ion homeostasis, II) ion gradients between plant tissues, and III) ion levels in plant mutants with compromised growth or heterogeneous phenotypes. - Highlights: • A cost-efficient TXRF procedure that enables precise, robust and highly reproducible measurements on plant tissue samples as small as 0.3 mg dry weight was developed overcoming the current limitations on sample amounts. • The procedure was verified by detecting the effect of environmental stress on plant ion homeostasis. • The potential of this method was shown by resolving for the first time developmental ion gradients between single plant leaves of A. thaliana and changes in the ionome of the recently discovered A. thaliana ion transport mutant, kea1kea2. • TXRF has high potential to contribute to a better understanding of plant physiology and to improve breeding programs and agriculture.