Nitrogen (N) is an essential element for all organisms, being a component mainly of proteins, amino acids, nucleic acids, membrane lipids, ATP, NADH. In plants, N promotes growth and development and is considered as a driving force for crop yield (Anas et al., 2020; Liu et al., 2022). This element is mainly absorbed from soil by the roots as nitrate or ammonium, although plants can absorb N from the leaves when applied by foliar spray (Xu et al., 2012). An adequate N availability in the root zone is crucial to obtain high yields (Tei et al., 2020). On the other hand, excessive N in cropping systems poses environmental hazards and may cause adverse effects on the crops themselves. From an environmental point of view, the main concerns are water eutrophication, soil acidification, and high rate of greenhouse gas emissions (Anas et al., 2020; Zamanian et al., 2024). The detrimental effects on crops include increased susceptibility to pathogen attacks (Sharma, 2020) and excessive vegetative growth (Leghari et al., 2016). Faster decay (Ierna et al., 2020) and deterioration of many quality parameters (Albornoz, 2016) are observed during postharvest. Nitrates, whose possible harmful effects on human health are known (Santamaria, 2006), are a major concern in leafy vegetable productions, especially for those species which are particularly prone to their accumulation (Cavaiuolo and Ferrante, 2014). Since excess nitrates accumulate in plant cell vacuoles, any factor affecting uptake and/or assimilation modulates their concentration in plant tissues.

The Non-Thermal Plasma (NTP) technology applied to crops can affect N metabolism in plants, including its accumulation (Brar et al., 2016; Peethambaran et al., 2015). The term “plasma” refers to a totally or partially ionized gas, composed of several chemically active components (Nehra et al., 2008). The NTP, belonging to plasmas artificially obtained by applying any form of energy to induce gas ionization (Nehra et al., 2008; Phan et al., 2017), can find scientific and industrial applications in various fields, including medicine, electronics, material sciences, agriculture, and food industry (Konchekov et al., 2023). In particular, NTP is trailed as a powerful technique for reducing chemical contaminants in agricultural products (Herianto et al., 2023). The main advantages are: the possibility of working with air, as it is at atmospheric pressure, low operating temperatures, short treatment times, efficient use of energy, and minimal environmental impact (Cherif et al., 2023; Rajan et al., 2023). On the other hand, some drawbacks limit the use of NPT, such as possible adverse alterations in the physicochemical characteristics of the treated food, and the difficulty to cover the entire surface during the decontamination treatments (Herianto et al., 2023). On the contrary, Plasma Activated Water (PAW), which is generated by treating water with a plasma stream, seems a more flexible alternative than the gaseous form for applications in the agricultural sector (Ruamrungsri et al., 2023). When energy input is applied to the gas, reactive oxygen and nitrogen species (ROS and RNS) are generated such as hydrogen peroxide, ozone, nitrate, nitrite, nitric oxide, ammonium ions. These molecules can be delivered to the gas-liquid interface and thus enrich PAW composition (Zhou et al., 2020). Moreover, other chemical aspects are modified in PAW such as a lower pH and a higher electrical conductivity (EC) and redox potential (Thirumdas et al., 2018).

In agriculture, plasma shows potentiality for pest and disease control due to its antimicrobial activity, but can also exert positive effects on plants like stimulating seed germination, root development, and plant metabolism, enhancing tolerance to biotic and abiotic stresses, improving nutrient availability and uptake, and increasing overall crop productivity and quality (Bora et al., 2022; Cannazzaro et al., 2021a, 2021b; Konchekov et al., 2023; Puač et al., 2018; Traversari et al., 2021). Regarding abiotic stress, NTP treatment has been showed to improve crop resistance to salinity (Veerana et al., 2024) and heavy metals (Ghasemzadeh et al., 2022). Although the mechanisms of plasma-plant interactions are not yet well understood, the use of plasma could be assimilated to that of a biostimulant, since it appears to be involved in the activation of various plant growth regulators and stress response genes, the induction of systemic resistance, and the strengthening of antioxidant defence mechanisms (Konchekov et al., 2023). Furthermore, PAW, administered as irrigation water, can directly counter pathogens that might attack the roots (Brandenburg et al., 2019) or the epigeal part (Cannazzaro et al., 2021a), and have a fertilizing effect due to the capacity of capturing atmospheric N (Puač et al., 2018). Among other valuable effects on plant quality, a significant increase in plant pigments and reduction of nitrate concentration in leafy vegetables irrigated with PAW have been observed (e.g., Cannazzaro et al., 2021a). Therefore, rocket, who naturally accumulates nitrates at the high levels (Cavaiuolo and Ferrante, 2014), has been chosen as test plant in this work. In particular, the wild rocket [Diplotaxis tenuifolia (L.) DC.] variety ‘Dragon’s Tongue’ has been selected for its flavour attributed to the presence of high anthocyanin and other compounds in the leaves, especially in the main leaf veins, which are red in colour.

Despite the potential of plasma technology for crop management, most studies have focused on seed or post-harvest treatments, while application during the cultivation phase has been scarcely investigated. The main difficulties concern the dosage and intensity of treatments, which are subject to dynamic changes also as a results of growing environment conditions. The aim of this work was to evaluate the effect of the interaction between NTP and N fertilization on crop yield and quality. For this purpose, the NTP was used to treat the nutrient solution supplied to wild rocket plants grown hydroponically in a greenhouse combining plasma treatment with different levels of N in the nutrient solution.

In this work the NTP treatment showed contrasting and interaction effects on rocket salad plants in relation to the N concentration in the nutrient solution. Three different levels of N were chosen representing a high, medium, and very low level of N. The first interesting N-dependent effect to note concerns plant yield. The NTP promoted the biomass accumulation at medium concentration (10 mM N), particularly at the 2^nd growing cycle that is representative of the marketing product stage, while caused detrimental effects on plant yield at the extreme concentrations (1 and 20 mM N). Indeed, NTP treatments have been found to improve biomass accumulation in several plant species (e.g., Cannazzaro et al., 2021b; Cui et al., 2019; Thirumdas et al., 2018). Despite this, the highest FW and DW were achieved by increasing N concentration, confirming that N fertilization is the prevalent factor involved in stimulating the biomass production. As it is well known, crop yield is indeed positively related to N availability in the root zone. However, when present at high concentration, this element may cause an excess accumulation of NO₃ in edible organs of leafy vegetables thereby making the product even unmarketable (Massa et al., 2018; Santamaria, 2006). Therefore, simple adding N fertilizers to increase yield may not be the best choice; instead, a proper agronomic strategy should be adopted to find the correct balance between produce yield and quality. Compared with other leafy vegetables, rocket accumulates much higher amount of NO₃ (Santamaria, 2006), a typical strategy developed by many wild species to counteract the negative effects of osmotic stresses (He et al., 2024). In lettuce, NTP limited the accumulation of NO₃ (Cannazzaro et al., 2021a). However, to the best of our knowledge, there is no work testing such effects on leafy vegetables under extreme, i.e., very low and very high, or otherwise variable N concentration in the root zone. Our results showed that there is a theoretical limit of N concentration in the root zone above which the NTP treatment would induce detrimental effects on plant physiology. This is likely due to an excess formation and accumulation, in the root zone, of oxidant molecules typically produced under NTP treatment, mainly belonging to the category of RNS, associated to the high level of N. In fact, the NTP-treated water may contain ROS and RNS (Thirumdas et al., 2018) that can be beneficial or harmful for plants, depending on their concentration (Cui et al., 2019). This negative effect of NTP was also observed in case of very long exposure or high intensity treatment of PAW (Cannazzaro et al., 2021a; Nicoletto et al., 2023; Than et al., 2022). Moreover, a higher N concentration in the nutrient solution supplied to rocket plants grown in soilless system has been shown to increase the peroxidase activity and malondialdehyde content, both indicators of oxidative stress (Villani et al., 2023). This suggests that elevated N levels might have had a synergic negative effect with the NTP treatment in promoting oxidative stress. Plants fertigated with 20 mM N and treated with NTP indeed showed a very strong reduction in biomass compared to untreated plants. This result coupled with a F_v/F_m reduction at the 2^nd growing cycle highlighting a possible toxic effect on the photosystem II, considering that chlorophyll fluorescence is one of the first parameters affected by oxidative stress (Moustaka and Moustakas, 2023). The negative effect on biomass and photosystem II may have been exacerbated by the cutting and thus more evident at the 2^nd productive stage.

A different mechanism appears to have contributed to the biomass reduction under low N. The dry biomass reduction found at 1 mM N in control plants was coupled with a strong increase in anthocyanins. This effect was optically visible by a red colouration of plants under this treatment (data not shown). Anthocyanins are involved in plant response to stress such as N deficiency through well characterized metabolic pathways (Li and Ahammed, 2023); thus, rocket plants not treated with NTP might be more strongly affected by low N level than the NTP treated plants. The stress in these plants might be suggested also by the increase in Mn concentration as a high concentration of this element is usually associated with a higher activity of the antioxidant system (Alejandro et al., 2020) in agreement with the higher anthocyanin content (Gould et al., 2002). The NTP treatment did not increase the concentration of anthocyanins, also under non-limiting N concentration, in contrast with previous results using a red variety of lettuce grown in similar hydroponic conditions in which an increase in these molecules was observed under treatment (Cannazzaro et al., 2021a). In our experimental conditions, the difference in FW between control and NTP treated plants was less evident than the difference in DW because NTP treated plants showed a general higher tissue hydration with lower DW percentage. This is a typical behaviour of plants exposed to NTP treatments, in which a reduced stomatal conductance would play a major role (Cannazzaro et al., 2021b). Nonetheless, NTP treatments has been suggested as a strategy to increase water use efficiency in cropping systems (Brar et al., 2016; Peethambaran et al., 2015), although such practice appears risky under operational conditions. Moreover, NTP treated plants showed a higher K concentration that might stimulate a greater turgor maintenance (Barker and Pilbeam, 2015). The higher DW percentage found in control plants at 1 mM N level compared to the NTP treated plants might be triggered also by the elevated anthocyanin content that has been showed to ensure higher fresh and dry weights under abiotic stress as nutrient deprivation (Yan et al., 2022). Despite the higher DW, control rocket salads had a strong decrease in N and other elements compared with NTP treated plants at 1 mM N. Indeed, the reddish coloration related to anthocyanin overproduction can be also a visual symptom of N deficiency (De Bang et al., 2021). The higher N concentration found in NTP treated plants might be related to the higher NO₃ ⁻ availability found in PAW, which would support the hypothesis that NTP treated water can, at least partially, replace conventional N fertilizers thus reducing their supply in agriculture (Ranieri et al., 2021; Thirumdas et al., 2018). However, in our experimental conditions NTP showed peculiar aspects related to the different N level. NTP treated plants increased Kjeldahl N concentration compared to the controls at 1 and 20 mM N and NO₃ concentration at 1 mM N while in the same plants a decrease in the dry biomass was observed. At the 2^nd harvest, all the investigated macrocations increased their concentration in plant tissues of N starved plants irrigated with PAW, which is consistent with the higher accumulation of macroanions (PO₄ and NO₃). Similar trends have been observed in the herbaceous organs of floricultural species except for Ca (Cannazzaro et al., 2021b; Traversari et al., 2021). Among the micronutrients, N fertilization had a negative effect on Zn concentrations on NTP untreated plants. Interestingly, this element decreased at increasing N levels while it was particularly high at 1 and 20 mM N levels under NTP treatment. Indeed, the positive effect of NTP treatment on Zn accumulation has been already found in begonia edible flowers (Traversari et al., 2021). The Zn increase particularly evident at 1 and 20 mM N might be also related to the higher level of oxidative stress associated to these NTP treatments as Zn has a well-recognised role in defence mechanisms involved in ROS response (Cakmak, 2000). Calcium, Mg, K, and Zn are essential elements in the human diet and their bioavailability is greater when taken through food rather than synthetic products. Therefore, many works are addressed to obtain biofortified vegetable products (Buturi et al., 2021; White and Broadley, 2009). At the operational level, the most practical and economic technique consists in the supply of high amount of fertilizers at high concentration of the target elements thereby causing their accumulation in edible plants. Fertigation, especially in soilless systems, shows a high efficiency and can be adopted for this purpose (Massa et al., 2023; Sambo et al., 2019). However, it should be highlighted that a massive use of chemical fertilizers may induce plant nutritional imbalance to the detriment of other nutrients. Moreover, above specific limit thresholds in the root zone the residual (unabsorbed) elements can accumulate in the system since the uptake of mineral elements is not proportional to the quantity supplied. These residuals may then accumulate in the environment or require disposal which has environmental and economic implications. Therefore, techniques and technologies that can stimulate the accumulation of nutrients in plants avoiding the use of fertilizers are indeed worth exploring.

In conclusion, the NTP treatment showed different effects related to the N concentration in the nutrient solution. At very low or high concentrations, it had negative effects on biomass. However, if considered the medium N concentration, NTP was able to improve plant biomass production and promote K and Zn accumulation in plant shoots suggesting its suitability for promoting yield and quality of rocket salad plants. Our results highlight also the importance to evaluate the threshold level of N for a crop to avoid the negative effects related to the oxidative stress. Therefore, NTP use is recommended only under an appropriate level of N in nutrient solution which should be carefully evaluated to avoid possible negative effects.