Nitrogen supply is one of the most common limiting factors for plant growth and development (Weisler et al., 2001; Das and J, 2015; Bouchet et al., 2016; Yadav et al., 2017), due to its essential role as a substrate for the biosynthesis of many vital cell components such as nucleic acids, amino acids, the photosynthetic pigment chlorophyll, and many more (Haynes, 1986; Budil and Thurnauer, 1991; Hawkesford et al., 2012; Wen et al., 2019; Zhang et al., 2019). Insufficient availability of N to plant cells results in shortage of these key metabolites, restricted cell metabolism, and thereby a reduction in most plant processes and developmental delays. It is therefore not surprising that N supply considerably affected the physiological and morphological status of the cannabis plants tested.

Nitrogen supply positively correlated with concentrations of photosynthetic pigments in the leaves (Figure 8), and with net photosynthesis rates (Figure 6A) up to 160 mgL⁻¹ N supply. N is a major constituent in these pigments, i.e., each chlorophyll molecule contains four atoms of N (Budil and Thurnauer, 1991). As was demonstrated before for numerous plant species (Evans, 1989; Dordas and Sioulas, 2008; Najm et al., 2012), in cannabis as well, the increase in N supply supported enhanced pigment production. Since one of the major factors influencing the rate of photosynthesis is the concentration of photosynthetic pigments (Emerson, 1929; Frank et al., 1991; Maekawa and Kokubun, 2005; Ionescu et al., 2009), we suggest that the low rate of photosynthesis under 30–80 mgL⁻¹ N reflects the low concentration of these pigments (Figure 6A).

Moreover, N supply is known to affect proteins of the Calvin cycle and thylakoids, which represent the majority of leaf N (Evans, 1989), forming a link between leaf N concentration and leaf CO₂ assimilation (Sinclair and Horie, 1989; Sung et al., 2013). The relation between leaf N, chlorophyll concentration, and photosynthesis is apparent in our results for low N supply—up to 160 mgL⁻¹ N. Further increase in N supply and concentrations of photosynthetic pigments at the 240–320 mgL⁻¹ N range were not accompanied by further increase in photosynthesis, since at those levels the photosynthetic pigments were no longer the limiting factor. Rather, the decline in photosynthesis under high N supply resulted from stomatal closure, which was accompanied by further reduction in stomatal conductance, transpiration rate, and intercellular CO₂ (Figure 6).

Another parameter affected by concentrations of the photosynthetic pigments is leaf color (Figure 1). The chlorotic-pale green-yellowish leaves of the low N treatments reflect the low levels of chlorophyll, while the dark green leaves at the 160–320 mgL⁻¹ N range indicate a sufficient level of pigments. Moreover, the similar leaf color at the 160–320 mgL⁻¹ N range strengthens the conclusion that at this range the pigments are no longer the limiting factor for carbon fixation, as already under 160 mgL⁻¹ N the impact of the pigments and the rate of photosynthesis are at their maximum (Figure 6A).

Photosynthesis is the main process governing plant production and growth, as photosynthesis rate indicates on carbohydrate production, and consequently biomass production and accumulation (Pearcy et al., 1981; Whitmarsh and Govindjee, 1999; Malhi et al., 2015). The response of the cannabis plant growth (e.g., plant height, stem width and side branches length; Figure 3) and biomass accumulation into the plant organs (Figures 2A–C) to N supply reflects the photosynthetic rate of the plant (Figure 6A), emphasizing the interplay between plant metabolism, energy status, and plant growth. The effect of N supply on plant development was visibly apparent (Figure 1) with optimal leaf, shoot, and root development identified under 160 mgL⁻¹ N, and impaired growth under higher and lower N supply. Unlike other growth parameters, node formation was not affected by the wide range of N supply tested in the present study (Figure 3B), demonstrating insensitivity to N availability. In other plants, node formation was indeed demonstrated to be regulated by other factors, primarily temperature, rather than by N supply (Ingram and Hilton, 1986; Berghage, 1998; Gabryszewska, 2011; Peart and Shoup, 2018).

As a prime macro-mineral that is utilized in large quantities by all plant organs, N is known for its impact on uptake and transport of other plant mineral nutrients by the roots (Miller et al., 1976; Hegde, 1987; Cimrin et al., 2004; Shivay et al., 2007; White, 2012; Savvas et al., 2018), which may lead to changes in ion transport and translocation in the plant. Although total N and N-NO₃ accumulation in the roots and stem increased with N supply throughout the N range tested, in the leaves, only N-NO₃ concentration increased with N supply while the total N was not affected by N levels greater than 160 mgL⁻¹ (Figures 4A,B). Total N is composed mainly of N-containing organic metabolites which compose the largest N reservoir in the leaf, as demonstrated before for other plant species (Evans, 1989; Sinclair and Horie, 1989), and N-NO₃ is only a small fraction of this reservoir. The results therefore teach us that under N supply > 160 mgL⁻¹ there was a decline in production and accumulation of N-containing organic metabolites in the leaves (Figures 4A,B). This decline in N-containing organic metabolites is in accord with the plant overall toxicity response to oversupply of N, as will be discussed later.

The optimum-shape response curves to increased N inputs that were obtained for the majority of macronutrients (P, K, Ca, Mg; Figure 4) is quite different from the response we reported before for K supply (Saloner et al., 2019), and can be correlated to the plant vitality status, i.e., high nutrient uptake and accumulation which are energy-consuming processes, reflect prime functional morpho-physiological status (White, 2012). Another worth-noting trend is that lower N supply promoted accumulation of metals such Mn, Ca, and Zn in the plant (Figures 4E, 5A,B), while Fe declined under the lower N supply (Figure 5C), demonstrating specificity in the response of ion uptake mechanisms to N supply. Enhanced supply of specific ions to the root zone can affect uptake and accumulation of other nutrients in the plant by competition for uptake. NH₄ is well known to compete with other cations such as Mg, Mn, and Ca (Geraldson, 1957; Cox and Reisenauer, 1977; Rayar and van Hai, 1977; Van Beusichem et al., 1988; White, 2012), and this was apparent also in the present study as the levels of these minerals reduced with the increase of N supply over 160 mgL⁻¹ (Figures 4E,F, 5B).

The osmotic potential response to N-input reflects trends of nutrient accumulation in the leaves (Figure 7B). Since N and N-NO₃ concentrations were greatly elevated with the increase in N supply, while concentrations of most other macronutrients were not highly affected, the increase in osmotic potential with N and N-NO₃ supply correlates with their accumulation in the tissue sap. The difference in trends between the osmotic potential curve that increases throughout the N concentration gradient, and the RWC curve (Figure 7A), which displays a maximum curvature, with drier leaves in both extreme N treatments, supports that the increase in osmotic potential is due to specific accumulation and not solely a result of tissue drying. This notion is supported by results of bean to N supply (Guo et al., 2007).

In agricultural systems, the plant is considered a production unit, and in this respect, it is vital to examine its efficiency. Since elevated N supply promoted a better functioning photosynthetic system, due to the high content of photosynthetic pigments, stomatal conductance could be reduced (Figure 6C). This decline diminished water loss by transpiration (Figure 6B), but at concentrations higher than 160 mg L⁻¹ also decreased the intercellular CO₂ concentration (Figure 6D). The significant reduction in transpiration and hence water loss (Figure 6B) made the plant more efficient in terms of water use (Figure 7C). Despite the considerable increase in water use efficiency, N use efficiency declined as N supply increased (Figure 2D), meaning that despite the higher biomass production under the 160 mgL⁻¹ N level, the plants produced less biomass per N unit. Hence, the plants are less efficient in terms of N use at the higher N levels. As nitrate was elevated in the roots with the increase in N supply (Figure 4B), it is presumed that the decrease in plant N use efficiency is due to a restricted nitrate reduction in the plant roots. Restricted ability to reduce nitrate in the roots may result from a feedback inhibition of nitrate reductase from the NH₄⁺ supplied and from ammonia-derived metabolites, as was demonstrated for many plants before (Srivastava, 1980; Konnerup and Brix, 2010; Piwpuan et al., 2013), or from a inactivity of nitrate reductase in roots, and this issue should be further investigated.

The decline in metabolism and phenotypic development under high N supply (240 mgL⁻¹ N and above) in the cannabis plant is the outcome of two main damage avenues; namely, specific detrimental effects of the nitrate or ammonium ions, and non-specific negative effects of high concentrations. Nitrate and ammonium, like any nutrient ions, have optimal concentration range in plant tissues in terms of plant response to N dose (Broadley et al., 2012; Hawkesford et al., 2012). When nitrate level exceeds the threshold of tolerable level (i.e., the toxicity threshold), it can induce cell damage by various mechanisms. First, nitrate assimilation elevates intracellular pH, because nitrate reduction to nitrite is a proton-consuming process that enriches the cytosol with OH^– (Britto and Kronzucker, 2005). This elicits enhanced production of organic acid anions to stabilize cell pH and maintain the anion–cation biochemical balance, but has a downside as well since the organic acid biosynthesis is energy-consuming and may dwindle energy availability in the cells thus contributing to growth restriction (Hauck et al., 1984). Furthermore, excess nitrate accumulation can be toxic as it results in nitrite and nitric oxide production—two molecules which are highly toxic to plants when over-accumulate (Hauck et al., 1984; Lamattina et al., 2003; Anjana and Iqbal, 2007; Zheng et al., 2016). As NH₄ supply was elevated as well at the high N treatments, reaching up to 58 mgL⁻¹ N-NH₄, it cannot be excluded that the detrimental effects were caused by NH₄ overdose, a well known and discussed phenomenon in plant nutrition (Hauck et al., 1984; Bernstein et al., 2005; Hawkesford et al., 2012). C. sativa responses to NO₃/NH₄ ratio, and the involved mechanisms are yet unknown and are currently under investigation in our laboratory. A non-specific effect of N toxicity may involve a non-specific concentration-driven osmotic stress in the plant tissues, i.e., a salinity effect. Such an effect is apparent from the rise in osmotic potential of the leaf tissue sap (with the increase in N supply and N-NO₃ concentration in the leaves; Figures 4B, 7B). The results also point at involvement of oxidative damage under N access and deficient conditions. Various abiotic stress conditions, including mineral deficiencies and toxicities, and N nutrition in particular, are accompanied by oxidative stress, i.e., unregulated increase in concentration of reactive oxygen species (ROS) in the plant tissue (Kong et al., 2013; Chokshi et al., 2017; Choudhury et al., 2017; Bellegarde et al., 2019). N deficiency and toxicity levels correlated in the present study with reduced membrane integrity, e.g., increased membrane leakage, that reflects oxidative damage and especially lipid peroxidation (Liu and Huang, 2000; Bernstein et al., 2010; Kravchik and Bernstein, 2013; Teissie, 2017) suggesting a role of oxidative stress in the deficiency and toxicity responses.

Nitrogen overdose often causes excessive production of organic N molecules as was discussed above, consuming high amounts of metabolic energy, which reduces energy and carbohydrates availability, and delays biomass production. Furthermore, N uptake and assimilation are energy-consuming processes which increase under high N supply (Mengel and Viro, 1978; Zapata et al., 1987; Huppe and Turpin, 1994; Hawkesford et al., 2012) and further dwindle energy availability. Ion uptake, that is an energy consuming process, indeed correlated in the present study with carbon fixation, and a decline in uptake and accumulation of vital nutrients P, Ca, Mg, and Mn took place at the high N treatments (Figures 4D–F, 5B).