The population inhabiting the Earth is predicted to reach almost 10 billion by 2050 (Taiz, 2013). The agriculture sector is, therefore, asked to increase the production of staple foods in order to satisfy the request of the growing population (Tilman et al., 2011; Kagan, 2016). However, due to soil degradation, the enhancement of agricultural yields could be necessarily obtained by intensifying production rather than extending cultivated areas (Lal, 2015; Kopittke et al., 2019). Although in the past decades the yield increase of key crops has been achieved by raising the use of agrochemicals (i.e., fertilizers and pesticides/herbicides), such massive applications, especially of N-based fertilizers, have posed severe environmental risks (Tilman et al., 2002). Indeed, the effectiveness of conventional fertilizers is threatened by their limited nutrient use efficiency (NUE), i.e., the ability of plants to acquire nutrients at root level and then to allocate them to the shoot and other sink organs. In general, low NUE values originate from fertilization rates that are higher than the ability of plants to take up nutrients (Guo et al., 2018). At present, for instance, it has been estimated that the average NUE for N-based fertilizers is approximately 50%, thus implying that half of the total N applied is not used by crops and lost in the environment (Lassaletta et al., 2014). To overcome these issues, several solutions have been adopted, for instance, soilless cultivation (Sambo et al., 2019). However, the development of new types of fertilizers might also represent a suitable approach to help intensify the agriculture production in a sustainable manner. In this context, the exploitation of nanotechnologies for the delivery of nutrients to crops can be envisaged as an interesting novel solution (Kopittke et al., 2019).

Nanotechnology offers the possibility of creating nanoparticles characterized by smaller size (typically below 100 nm) with respect to bulk materials, and by both high surface area and high reactivity (Ullah et al., 2020). These characteristics open a wide range of opportunities for the development of nanofertilizers featuring higher effectivity, lower ecological risks, and lower economics costs, as compared to their traditional counterparts (Ullah et al., 2020). Interestingly, the use of nanomaterials as carriers will allow tuning the release of the fertilizers in a controlled manner. Such slow release will indeed help prolonging the nutrient persistence in the agro-ecosystem and providing crops with optimal nutrients levels for a prolonged period of time, thus resulting in higher NUE (Guo et al., 2018). According to Kopittke et al. (2019), a wide range of nanomaterials can serve as fertilizers in agriculture, thereby including those composed of the nutrient to be delivered as well as those loaded with the nutrient(s) of interest. In this context, a great attention has been given lately to nanocrystals of hydroxyapatite [HA, Ca₅(PO₄)₃OH] (Kopittke et al., 2019). Nanosized hydroxyapatite presents a high surface area to volume ratio and also the potential of releasing macronutrients, namely, calcium (Ca) and phosphorus (P), for fertilization purposes (Guo et al., 2018). According to these characteristics, HA-based nanoparticles have been studied to improve, on one hand, P nutrition in plants (Liu and Lal, 2014; Xiong et al., 2018; Kopittke et al., 2019; Sega et al., 2019) and, on the other hand, as a carrier to deliver other important macronutrients, such as N (Kottegoda et al., 2011, 2017; Madusanka et al., 2017). Nevertheless, in spite of the importance of N as a plant macronutrient and its relevance for crop productivity, very few studies have been published on the topic so far (Kopittke et al., 2019). First evidence gathered on urea-modified HA nanoparticles showed that the nanofertilizer was effective in releasing urea over a period of 60 days, in contrast with the rapid solubilization of the traditionally used bulk urea (Kottegoda et al., 2011), ensuring a 50% increase in the yield of Oryza sativa plants, as compared to controls (Kottegoda et al., 2017). More recently, amorphous calcium phosphate (ACP) nanoparticles, have been developed and doped with urea (U-ACP) (Ramírez-Rodríguez et al., 2020a; Carmona et al., 2021). Similarly to urea modified HA, U-ACP showed a controlled release of macronutrients in soil over time and, most importantly, allowed a wheat grain yield comparable to that of control plants, albeit the N dose applied with U-ACP has been halved as compared to controls (Ramírez-Rodríguez et al., 2020a,b). Interestingly, U-ACP also showed to impact the quality of agricultural products. In Tempranillo grapevine plants, the foliar application of a U-ACP suspension, conveying a N fertilization rate reduced by about 15 times as compared to traditional viticulture practice, induced an increase in yeast-assimilable nitrogen (YAN) and in amino acid concentration at berry level, thus demonstrating a positive impact on the fermentability of grapes (Pérez-Álvarez et al., 2021). More recently, a 2-year study carried out on Pinot gris grapevines highlighted that the fertilization practice with U-ACP, at a 20% lower N fertilization rate as compared to traditional practices, did not alter the growth parameters of plants, as well as, the yield and its quality parameters (Gaiotti et al., 2021). In this regard, the aromatic profile analyses of berries collected from U-ACP fertilized plants did not present any significant alterations as compared to those produced by urea-fertilized samples (Gaiotti et al., 2021). This result clearly suggests that U-ACP can be a valid alternative to bulk urea with undoubted advantages from both environmental and economic standpoints. However, despite these pieces of evidence, information about the physiological and molecular aspects underpinning their action is still scarce.

Bulk urea represents the most widely used fertilizer in agriculture because of its economic convenience and high N content (Beier and Kojima, 2021). Plants have evolved quite a complex system to take up urea from the external environment that is based on channels, namely, nodulin 26-like intrinsic proteins (NIPs) and plasma membrane intrinsic proteins (PIPs), and on a specific transporter, degradation of urea (DUR) 3. Tonoplast intrinsic protein (TIP) channels have also been identified. While NIPs, PIPs, and TIPs seem to marginally contribute to urea uptake, and just in very specific conditions, DUR3 is accounted as the main carrier for this N form (Liu et al., 2003; Wang et al., 2012; Zanin et al., 2014). DUR3 transporter is localized in root cells plasma membrane, and it has been shown to act as a high affinity proton-dependent symporter (Liu et al., 2003). The transporter is encoded by the DUR3 gene (Kojima et al., 2007; Wang et al., 2012; Zanin et al., 2014), whose transcriptional modulation shows down-regulation in response to nitrate and ammonium supply, whereas N starvation and urea fertilization induce an up-regulation of DUR3 expression in several plant species (Beier and Kojima, 2021). These observations corroborate the existence of the so-called induction phenomenon, already demonstrated for nitrate uptake in plants. To this regard, Zanin et al. (2014) showed that maize plants, starved of N and afterward exposed to urea for 4 h, could take up the nutrient with a different kinetic behavior compared to untreated plants, thereby displaying a higher Km and Vmax. Indeed, such increase indicates a higher capacity of the transport system, yet features lower specificity (Siddiqi et al., 1990).

On these premises, the aim of this study was to investigate the physiological and molecular responses of Cucumis sativus L. (used a plant model for crops) to exposure to U-ACP. A traditional N fertilizer (i.e., bulk urea) has been used as control. By adopting a time-course experiment, the urea uptake dynamics at root level have been investigated by monitoring both urea acquisition rates and modulation of CsDUR3. In a mid-term experiment, on the other hand, plant growth parameters, the accumulation of N in both root and shoots, as well as the general ionomic profile of both tissues, have been analyzed to highlight possible synergism and antagonism between U-ACP and other mineral nutrients.

The quest for more sustainable approaches to meet the challenges of a growing world population is forcing agriculture toward the development of innovative fertilization practices (Tilman et al., 2011; Kagan, 2016). In this regard, an attractive solution might be represented by the application of agrochemicals based on nanomaterials, which can feature higher effectivity, lower ecological risks, and lower costs as compared to their traditional counterparts (Ullah et al., 2020). Previous studies have already demonstrated the suitability of nanoparticles based on amorphous calcium phosphate (ACP) nanoparticles as carriers of macronutrients (e.g., urea). In particular, it has been demonstrated that upon reduction of N fertilization rate, plants supplemented with ACP could mirror the same agronomic performance (i.e., yield and quality) of plants treated with bulk fertilizers (Ramírez-Rodríguez et al., 2020a,b; Carmona et al., 2021; Gaiotti et al., 2021; Pérez-Álvarez et al., 2021). Despite this evidence, the knowledge of plant physiological responses to urea-doped ACP (U-ACP) nanoparticles is still scarce.

The data hereby presented corroborate further that urea itself, as previously demonstrated for the inducible high affinity nitrate transport system (Orsel et al., 2002), can function as a signal for the induction of its own specific transport system in cucumber; indeed, the induced plants showed higher urea uptake rate and significant up-regulation in the expression of the CsDUR3 gene (Supplementary Figure 3 and Figure 1). To date, the kinetics of urea uptake mechanism in higher plants has only been characterized in Arabidopsis, rice, and maize (Kojima et al., 2007; Wang et al., 2012; Zanin et al., 2014). Interestingly, maize plants exposed for 4 h to 1 mM urea showed a twofold increase in substrate uptake rate (Zanin et al., 2014), which is consistent with the kinetics data obtained here in the time-course experiment (Supplementary Figure 3 and Figure 1). After the initial up-regulation, the uptake rate showed a decline that could resemble the de-induction phenomenon described in the case of NO₃^– acquisition at root level (Glass et al., 2001; Nikolic et al., 2012). The kinetics of urea uptake in cucumber plants was also supported by the modulation of the CsDUR3 gene, which was up-regulated at 8 HAT, as compared to the not induced plants, and then down-regulated at 24 HAT (Figure 1B). These results further expand the knowledge of urea uptake dynamics provided by previous studies on different plant species (Kojima et al., 2007; Wang et al., 2012; Zanin et al., 2014). In fact, they are in good agreement with the observations carried out on rice seedlings, in which OsDUR3 was induced 3 h after treatment with 1 mM urea (Wang et al., 2012). On the other hand, experiments carried out on A. thaliana and maize seedlings highlighted the down-regulation of DUR3 expression 3 and 6 h after the treatment with 1 mM urea (Kojima et al., 2007; Zanin et al., 2014). These observations might suggest a different control of the urea uptake machinery depending on plant species, as also well described for the transcriptional regulation of ammonium transporters (Goyal and Huffaker, 1986; Mack and Tischner, 1994; Kronzucker et al., 1998; Rawat et al., 1999; Sonoda et al., 2003; Loqué and von Wirén, 2004; Yuan et al., 2007; Pii et al., 2019). When the cucumber plants were treated with NP, regardless of the concentration level, the pattern of urea uptake rates showed, at the beginning, a similar induction trend as observed in the urea-treated ones, whereas it did not display a de-induction phase (Figure 1A), with CsDUR3 modulated accordingly (Figure 1B). The prolonged activation of the urea uptake machinery in cucumber plants treated with NP suggests the ability of these plants to acquire the nutrient for a longer period of time with respect to conventional treatments; this resulted, for instance, in the higher accumulation of N at root level, when a 7-day fertilization period was considered (Figure 4A). Assuming that urea could function as a signal for the induction of its own uptake system (Beier and Kojima, 2021), the acquisition and molecular data obtained from the present experimental system might be ascribable to the kinetics of urea release from nanoparticles. Indeed, it has been demonstrated that up to 90% of the urea loaded on nanoparticles is released within 1 h by U-ACP dispersed in an aqueous solution (Ramírez-Rodríguez et al., 2020a; Carmona et al., 2021). Notwithstanding, the residual urea is released with a slower kinetic, ranging from 24 h up to 1 week, accordingly with the nanoparticles dissolution kinetics (Ramírez-Rodríguez et al., 2020a; Carmona et al., 2021).

Although the cucumber plants fertilized with NP and NP.5×showed higher N accumulation in the roots as compared to both the control and urea-treated plants, N accumulation was higher in the shoots of plants supplied with bulk urea (Figure 4B). In fact, both the NP- and NP.5×-fertilized plants presented lower N content in shoots, albeit this drop was significant only in the case of the NP.5×treatment in comparison to bulk urea. The differences between bulk urea and U-ACP observed in the translocation and allocation at leaf level could be ascribable to the diverse chemical structure of the nutrient. Consequently, the pathways involved in the uptake, translocation, and assimilation mechanisms might also be different. Bulk urea, for instance, is 100% available when it is administrated to plants. On the other hand, when U-ACP nanoparticles are dispersed in an aqueous solution, a quick release of the majority of urea is observed and it is then followed by slow solubilization (Ramírez-Rodríguez et al., 2020a; Carmona et al., 2021). Moreover, recent evidence has also shown that U-ACP nanoparticles can penetrate the root epidermis and reach the central cylinder via an apoplastic movement, which should become symplastic to cross the Casparian strip (Pérez-de-Luque, 2017; Ramírez-Rodríguez et al., 2020b). It is also worth noting that the different uptake dynamics could not only be related to the timing of urea release from the nanoparticles, but they could also be due to the nanoparticles themselves. In fact, analog experiences studying Fe nutrition in tomato demonstrated that, depending on the chelating agent (phytosiderophores vs. citrate vs. water extractable humic substances), plants could take up and allocate the micronutrient with different efficiency, albeit Fe was supplied at the same concentration in the growth substrate (Tomasi et al., 2013). Therefore, considering the peculiarities of U-ACP with respect to bulk urea, it might not be surprising that in over a 7-day fertilization period, slight differences in the N content of shoots were observed among the treatments (Figure 4B). Nevertheless, it is interesting to underline that the cucumber plants treated with NP.5×performed better in terms of N acquisition with respect to both the NP and urea treated ones (Carmona et al., 2021).

As also already observed (Carmona et al., 2021), despite the differential accumulation of N in plant tissues, no significant alteration in the biomass accumulation at root and shoot levels as well as in leaf chlorophyll content has been observed in the cucumber plants according to the fertilization treatments (Figure 2).

Several pieces of evidence have demonstrated that the mineral composition of plant tissues is tightly related with different aspects of (i) the edaphic environment (such as the chemical and physical characteristics of the growth substrate) as well as (ii) agricultural practices (such as fertilization and the application of biostimulants, e.g., plant growth-promoting rhizobacteria). The ionomic profiling of plant tissues subjected to different fertilization practices can, for instance, highlight possible phenomena of synergism and/or antagonism between mineral elements, thus resulting in differential accumulation of essential and/or non-essential mineral nutrients (Nikolic et al., 2007; Tomasi et al., 2009; Pii et al., 2015; Astolfi et al., 2018; Kolega et al., 2020; Scagliola et al., 2021). The treatment of cucumber plants with NP, regardless of the concentration level, caused an increased accumulation of calcium (Ca) and phosphorus (P) at both root and shoot levels (Figure 5). It is, however, noteworthy that, by supplying NP in order to meet plant N nutrition requirements, neither P nor Ca reached critical concentration levels to produce toxicity symptoms in cucumbers, as also supported by the measurements of relative chlorophyll contents (Figure 2A) and by previous observations (Ramírez-Rodríguez et al., 2020b; Carmona et al., 2021; Gaiotti et al., 2021; Pérez-Álvarez et al., 2021). These data are coherent with the fact that the NP used in this experiments was essentially composed of Ca and P, and that several authors have previously used it as P fertilizers for crop plants (Liu and Lal, 2014; Marchiol et al., 2019). Indeed, P is an essential macronutrient for plants (Hopkins and Hansen, 2019); however, using the currently available fertilization approaches, only a minor part (not more than 10%) is actually used by plants, mainly because of low P use efficiency (PUE) (Kopittke et al., 2019). Such scenario is additionally worsened by the accumulation of heavy metals in agricultural soils, for instance copper (Cesco et al., 2021), which can interfere with the P uptake mechanisms, thus contributing to further limitation in the ability of plants to acquire the nutrient (Feil et al., 2020). In this context, the application of nanofertilizers characterized by the slow release of P (Liu and Lal, 2014; Carmona et al., 2020; Ramírez-Rodríguez et al., 2020a), alongside urea, could possibly allow for the increase of PUE in crops. On the other hand, the enhanced concentration of Ca in plant tissues can also represent a benefit brought about by NP-based nonofertilizers. Calcium is an essential element for both plants and animals, thereby playing different roles, from both structural and biochemical (i.e., signaling) points of view (Sharma et al., 2017). Being the major staple crops, poor sources of Ca, the application of fertilization strategies that lead to Ca fortification in the edible part of plants might indeed increase their nutritional value. In addition, recent evidence has also pointed out that an enhanced concentration of Ca in the plant tissue could be related to higher resistance against the infection of fungal pathogens (Cesco et al., 2020), since it plays a crucial role in the stabilization and strengthening of the cell wall (Elmer and Datnoff, 2014). On the other hand, the urea-treated plants showed a prominent accumulation of Mg, S, and Zn (Figure 5), which is consistent with data recently obtained from hydroponically-grown maize plants fully fertilized with bulk urea (Buoso et al., 2021a,b). Even though literature concerning the synergistic or antagonistic relationship between either urea or NP and other mineral nutrients is still lacking, the higher Zn accumulation observed in the urea-treated plants with respect to the NP-treated ones could be ascribed to Zn/P antagonism; indeed, for several plant species, the increased uptake of P has been shown to inhibit the ability of plants to take up Zn, often causing the appearance of Zn deficiency symptoms (Reed, 1946; Cakmak and Marschner, 1987; Broadley et al., 2010; Santos et al., 2021).

In conclusion, this research aimed to understand the physiological and molecular responses of urea uptake mechanism in the model plant C. sativus L. upon fertilization with innovative nanofertilizers based on urea-doped amorphous calcium phosphate nanoparticles (NPs). The data hereby obtained demonstrated that the slow release of urea from NPs and/or the chemical composition of NPs themselves could contribute to the up-regulation of the urea uptake system for a longer period as compared to plants treated with bulk urea. This prolonged activation was indeed mirrored by higher accumulation of N in the NP-treated plants, even when the concentration of urea conveyed was halved (i.e., NP.5×treatment), thus confirming the higher performance previously described (Carmona et al., 2021). However, the higher accumulation of N was only partially translated to a higher biomass production, at least in this experience. In addition, besides impacting N nutrition, NPs also enhanced Ca and P concentration in cucumber tissues, which might possibly affect plant growth and yield, and the nutritional value of the agricultural product. Nevertheless, future studies will be necessary to address the suitability of NP-based fertilization strategies for different crops cultivated in different soil types under open field conditions.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

NM, FG, SC, and YP: experimental design. SF, GR, MA, and FC: experiment execution. SF, GR, MA, and YP: data collection. SF, GR, MA, FC, and YP: data analyses and visualization. SC and YP: data interpretation. SF, FG, SC, and YP: manuscript writing and critical revision. NM and YP: financial support. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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