Synchrotron-based X-ray analysis of living wheat leaves showed that foliar-applied ZnEDTA is taken up as a ligand complex, and typical Zn agricultural application rates may induce localized toxicity

In sunflower, both the trichomes and cuticle appear to be important for foliar Zn absorption.

No abstract

Environmental contextZinc, an essential micronutrient often applied to crops as a fertiliser, can be difficult to analyse in plants due to limitations of conventional techniques. Here, we use radiotracers and a non-destructive imaging technique to visualise how zinc applied as a nanofertiliser moves within wheat plants over time. This is an important step towards developing cost-effective fertilisers to help solve one of the world’s most widespread plant deficiencies. AbstractZinc (Zn) deficiency affects half of the world’s arable soil and one-third of the world’s human population. Application of Zn foliar fertilisers to cereal crops can be an effective way to increase grain Zn content; however, commonly used formulations can scorch the leaf (e.g. soluble Zn salts) or are prohibitively expensive (e.g. chelated Zn, ZnEDTA). Zinc oxide nanoparticles (ZnO-NPs) may offer an efficient and cost-effective alternative, but little is known regarding the mechanisms of Zn uptake and translocation within the plant. Foliar-applied Zn is analytically challenging to detect, locate and quantify, as it is omnipresent. Furthermore, any single analytical technique does not have the detection limit or spatial resolution required. In this study, the uptake and mobility of foliar-applied ZnEDTA, ZnO-NPs and ZnO microparticles (ZnO-MPs) to wheat (Triticum aestivum L.) were investigated using inductively coupled plasma mass spectroscopy (ICP-MS), synchrotron-based X-ray fluorescence microscopy (XFM) and radiotracing techniques using 65Zn-labelled formulations. The three techniques were compared to highlight limitations and advantages of each. We also report, for the first time, a novel time-resolved invivo autoradiography imaging technique that can be used to visualise 65Zn in live plants treated with foliar applications of 65ZnO-NPs and MPs. The images were supplemented by gamma spectroscopy analysis for quantification. The results of this study provide important insights into the analytical challenges faced when investigating foliar-applied Zn nanofertilisers in plants. Potential solutions using nuclear techniques are also discussed, which in turn may ultimately lead to the development of more efficient foliar fertilisers.

Foliar absorption of zinc (Zn) is limited by several barriers, the first of which is the leaf cuticle. In this study, we investigated the absorption of Zn from Zn oxide nanoparticles (ZnO-NPs) in wheat (Triticum aestivum cv Gladius) and sunflower (Helianthus annuus cv Hyoleic 41) to determine the importance of NP surface coating for Zn absorption. Fourier transform infrared (FTIR) spectroscopy showed a higher polysaccharide content in the wheat cuticle than sunflower, indicated by a more pronounced glycosidic bond at 1020 cm −1 , but wax and cutin content were similar. Scanning electron microscopy (SEM) revealed that trichome density was twice as high in wheat (3600 AE 900 cm −2) as in sunflower (1600 cm −2) and stomatal density four times higher in sunflower (6400 AE 800 cm −2 in wheat and 22 900 cm −2 in sunflower). Suspensions of ZnO-NPs with coatings of different hydrophobicity were applied to leaves to compare Zn absorption using X-ray fluorescence microscopy (XFM) and inductively coupled plasma mass spectroscopy (ICP-MS). Absorption of Zn was similar between wheat and sunflower when Zn was applied at 1000 mg Zn l −1 , but much less Zn was absorbed from all ZnO products than from soluble Zn fertiliser. Particle coating did not affect Zn absorption, but it may facilitate particle adhesion to leaves, providing a longer-term source of resupply of Zn ions to the leaves. Differences in leaf surface characteristics did not affect Zn absorption, indicating that the cuticle is the main pathway of absorption under these conditions.

Using zinc (Zn) foliar fertilizers to enhance the grain quality of wheat (Triticum aestivum) can be an effective alternative or supplement to Zn soil fertilizers. However, knowledge about the mechanisms of Zn absorption and translocation following foliar application is scarce. Here, autoradiography and γ-spectrometry were used to investigate the behavior of 65Zn applied to wheat leaves as soluble 65Zn chloride (65ZnCl2), chelated 65Zn (65ZnEDTA), 65Zn oxide nanoparticle (65ZnO-NP) suspensions, and 65ZnO microparticle (65ZnO-MP) suspensions. The largest amount of 65Zn absorption occurred in 65ZnCl2 treated leaves. However, this treatment (65ZnCl2) also had the lowest proportion of absorbed 65Zn translocated away from the treated leaf after 15 d due to leaf scorching (p = 0.0007). Foliar-applied 65ZnO-NPs and 65ZnO-MPs had the lowest absorption, but 65ZnO-NPs had the highest relative translocation. 65Zinc EDTA was intermediate, with higher 65Zn absorption than 65ZnO treatments but similar translocation. Regardless, the majority of the foliar-applied 65Zn remained in the treated leaf for all treatments. Furthermore, 65ZnO-NPs and 65ZnO-MPs accumulated in plant nodes, suggesting that Zn was absorbed as dissolved 65Zn and particulate 65ZnO. Overall, the form and amount of absorbed 65Zn affected translocation.

Foliar zinc (Zn) fertilization is an important approach for overcoming crop Zn deficiency, yet little is known regarding the subsequent translocation of this foliar-applied Zn. Using synchrotron-based X-ray fluorescence microscopy (XFM) and transcriptome analysis, the present study examined the translocation of foliar absorbed Zn in sunflower (Helianthus annuus) leaves. Although bulk analyses showed that there had been minimal translocation of the absorbed Zn out of the leaf within 7 days, in situ analyses showed that the distribution of Zn in the leaf had changed with time. Specifically, when Zn was applied to the leaf for 0.5 h and then removed, Zn primarily accumulated within the upper and lower epidermal layers (when examined after 3 h), but when examined after 24 h, the Zn had moved to the vascular tissues. Transcriptome analyses identified a range of genes involved in stress response, cell wall reinforcement, and binding that were initially upregulated following foliar Zn application, whereas they were downregulated after 24 h. These observations suggest that foliar Zn application caused rapid stress to the leaf, with the initial Zn accumulation in the epidermis as a detoxification strategy, but once this stress decreased, Zn was then moved to the vascular tissues. Overall, this study has shown that despite foliar Zn application causing rapid stress to the leaf and that most of the Zn stayed within the leaf over 7 days, the distribution of Zn in the leaf had changed, with Zn mostly located in the vascular tissues 24 h after the Zn had been applied. Not only do the data presented herein provide new insight for improving the efficiency of foliar Zn fertilizers, but our approach of combining XFM with a transcriptome methodological system provides a novel approach for the study of element translocation in plants.