Unveiling foliar uptake and assimilation pathways of manganese oxide nanoparticles through multimodal bioimaging

Manganese (Mn) is an essential plant micronutrient serving key functionalities in several physiological processes. Mn availability in the soil is highly regulated by soil parameters such as soil pH and redox status. Especially in calcareous and porous, sandy soils soluble Mn is rapidly oxidized and converted into plant-unavailable Mn(IV) oxides (Mn(IV)Os). Prolonged Mn scarcity can severely impact plant growth and result in yield losses. To prevent or alleviate Mn deficiency in crops, Mn is commonly applied via foliar fertilization. Despite being rapidly assimilated by plants, Mn is a phloem immobile nutrient and as such, it does not remobilize from old to newly developing organs. Consequently, repeated foliar Mn applications are required during the crop growing season to limit Mn-deficiency, representing a timeconsuming, costly and unsustainable approach.

The aim of this PhD was to develop a NP-based system that allows for efficient Mn delivery to plants via foliar fertilization and provide mechanistic insights regarding uptake, distribution and assimilation of Mn NPs in plants. The first objective was to synthesize and characterize Mn NPs suitable for foliar application experiments. Poly-acrylic acid-coated Mn(II)O (PAAMnO) NPs were chosen as candidate for testing on plants. PAA-MnO NPs showed pH- responsive release of Mn at physiological relevant in vitro settings and remarkable stability within a broad range of concentrations, making them promising candidates for foliar nanofertilization. Modified PAA-MnO NPs incorporating fluorescent dye and tracer elements were also synthesized to allow for NP detection and bioimaging in plants.

The second objective consisted in describing the NP uptake pathways, in planta NP dissolution dynamics and potential translocation out of the exposed leaves, using a combination of bioimaging and analytical techniques in barley plants. Confocal laser scanning microscopy (CLSM), nano-computed tomography (nano-CT) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) revealed that PAA-MnO NPs enter the leaf via stomata within a few hours after application. Following uptake, LA-ICP-MS on barley leaf cross-sections showed that NPs localize in proximity of and possibly within the leaf vasculature, where they may start dissolving. Furthermore, LA-ICP-MS highlighted differences in uptake and mesophyll distribution between foliar-applied NPs and naked ions. ICP-MS was applied to study NP dissolution and mobility inside the plant, revealing that NPs accumulated in the exposed leaf, where they released Mn ions, and promoted only marginal Mn remobilization within the four-day period following foliar application.

The third objective involved testing PAA-MnO NPs on Mn-deficient barley plants to evaluate their effects on the Mn metabolic functionalities. Imaging-PAM chlorophyll a fluorescence and inductively coupled plasma-mass spectrometry (ICP-MS) were used to assess leaf uptake and assimilation of NPs in comparison with conventional Mn sources (i.e. MnSO4 and MnCl2). Furthermore, particular emphasis was placed on optimizing NP formulation properties and assessing their influence on leaf NP uptake efficiency. Data obtained from the Imaging-PAM assay shows that PAA-MnO NPs were as effective as soluble salts at restoring Mn-functionality in photosynthesis. Notably, NPs did not induce leaf scorching as opposed to Mn soluble salts. The assay also indicated that ionic Mn rapidly entered the leaf xylem and remobilized towards the leaf tip promoting Mn restoration in a broader leaf area, whereas PAA-MnO NPs induced only local Mn restoration within 72 hours. ICP-MS analysis showed that the addition of a humectant in the NP formulation is required to promote NP uptake in barley leaves.

Overall, the set of methods utilized in the current study allowed for a comprehensive investigation of the interactions and effects of Mn NPs on Mn-deficient barley plants. The combination of bioimaging techniques highlighted the importance of stomata structures in the uptake of PAA-MnO NPs in barley. Furthermore, they showed that NPs can restore Mn- functionality in photosynthesis, but also that ionic Mn treatments are taken up more efficiently allowing for a broader Mn restoration in exposed leaves. We have also shown that optimal formulation properties are key to promote NP uptake in barley plants. While further research is required to clarify the mechanisms preventing PAA-MnO NPs remobilization, the attractive properties of NPs that enable leaf penetration and slow Mn release inside the apoplast underline the potential of nanocarriers for the foliar delivery of Mn. Thus, it is hoped that the knowledge here produced will be extrapolated to design more efficient Mn NPs capable of remobilizing prior to dissolution and advance sustainable foliar Mn fertilization.

Finally, part of this PhD was dedicated to writing a review article illustrating the basic principles and fundamental aspects of foliar nanofertilization (Paper I – Chapter 5) and a commentary paper (Paper II – Chapter 6) highlighting common shortcomings and misinterpretations found in phyto-nanotechnology.