X-ray fluorescence spectroscopy (XRF) is an analytical tool used to determine the elemental composition in a myriad of sample matrices. Due to the XRF non-destructive feature, this technique may allow time-resolved plant tissue analyses under in vivo conditions, and additionally, the combination with other non-destructive techniques. In this study, we employed handheld and benchtop XRF to evaluate the elemental distribution changes in living plant tissues exposed to X-rays, as well as real-time uptake kinetics of Zn(aq) and Mn(aq) in soybean (Glycine max (L.) Merrill) stem and leaves, for 48 hours, combined with transpiration rate assessment on leaves by an infrared gas analyzer (IRGA). We found higher Zn content than Mn in stems. The latter micronutrient, in turn, presented higher concentration in leaf veins. Besides, both micronutrients were more concentrated in the first trifolium (i.e., youngest leaf) of soybean plants. Moreover, the transpiration rate was more influenced by circadian cycles than Zn and Mn uptake. Thus, XRF represents a convenient tool for in vivo nutritional studies in plants, and it can be coupled successfully to other analytical techniques.

In vivo and micro chemical analytical methods have the potential to improve our understanding of plant metabolism and development. Benchtop microprobe X-ray fluorescence spectroscopy (μ-XRF) presents a huge potential for facing this challenge. Excitation beams of 30 μm and 1 mm in diameter were employed to address questions in seed technology, phytopathology, plant physiology, and bioremediation. Different elements were analyzed in several situations of agronomic interest: (i) Examples of μ-XRF yielding quantitative maps that reveal the spatial distribution of zinc in common beans (Phaseolus vulgaris) primed seeds. (ii) Chemical images daily recorded at a soybean leaf (Glycine max) infected by anthracnose showed that phosphorus, sulfur, and calcium trended to concentrate in the disease spot. (iii) In vivo measurements at the stem of P. vulgaris showed that under root exposure, manganese is absorbed and transported nearly 10-fold faster than iron. (iv) Quantitative maps showed that the lead distribution in a leaf of Eucalyptus hybrid was not homogenous, this element accumulated mainly in the leaf border and midrib, the lead hotspots reached up to 13,400 mg lead kg^-1 fresh tissue weight. These case studies highlight the ability of μ-XRF in performing qualitative and quantitative elemental analysis of fresh and living plant tissues. Thus, it can probe dynamic biological phenomena non-destructively and in real time.

In this study, we characterized Zn seed coats adhered on soybean seed surface [Glycine max (L.) Merrill] and then investigated the effects of such coatings on germination and finally monitored the transference of Zn from the coatings to the seedlings. For that, seeds were coated with ZnSO₄, 40 nm ZnO, and 5 µm ZnO at 4 mg of Zn per kg of seed. The shape and homogeneity of Zn deposits on the seed surface depended on the type of Zn source. X-ray spectroscopy unraveled that before the germination, Zn neither crossed the seed coat nor was biotransformed. Regardless of the particle size, ZnO-based treatments increased germination ratio, seedlings root, and shoot development, whereas ZnSO₄ suppressed them. After sowing, most of Zn on the seed coat was transferred to soil, while less than 13 wt% was assimilated by the seedling 14 days after sowing. Either this minor fraction of Zn was taken up during the imbibition process or absorbed by during seedling growth. Zinc oxide-based treatments represent a viable alternative to Zn supply in order to boost soybean germination and seedling development.