Nitrogen Source Matters: High NH 4/NO 3 Ratio Reduces Cannabinoids, Terpenoids, and Yield in Medical Cannabis

Abstract

Introduction

Cannabis (Cannabis sativa L.) is used by humanity as a medicinal crop since ancient times. In recent years, due to increasing awareness of the plants’ potential for modern medicine, its cultivation is spreading worldwide for the rapidly evolving medical cannabis industry (Decorte and Potter, 2015; Chouvy, 2019). Drug-type medical cannabis plants yield inflorescences rich in hundreds of phytochemicals such as cannabinoids, terpenoids, and flavonoids, which are the source of the plants’ biological activity (Russo et al., 2003; Andre et al., 2016; Shapira et al., 2019). The biosynthesis of these secondary metabolites is affected by environmental and cultivation conditions (Magagnini et al., 2018; Bernstein et al., 2019a,2005; Danziger and Bernstein, 2021a,b; Rodriguez-Morrison et al., 2021; Westmoreland et al., 2021); and the increasing demand by the pharmacological industry for a high-quality chemically standardized plant product requires understanding of the plant physiological and metabolic responses to exogenous factors, which is very limited today.

We have recently identified a sensitivity of cannabinoid and terpenoid production in drug-type (medical) cannabis to mineral nutrition, including N (Saloner and Bernstein, 2021) and P status (Shiponi and Bernstein, 2021b), and humic acid supplementation (Bernstein et al., 2019b). Progress was also made in understanding the nutritional requirements of the cannabis plant at the vegetative phase of development (Saloner et al., 2019; Saloner and Bernstein, 2020; Shiponi and Bernstein, 2021a), plant response to organic fertilization and interaction between nutrients (Caplan et al., 2017; Bevan et al., 2021), plant architectural manipulation (Danziger and Bernstein, 2021b,c), and drought stress (Caplan et al., 2019). The current study was undertaken to study the effects of N assimilation from an ammonium (NH₄⁺) vs. nitrate (NO₃^–) N source on medical cannabis morpho-development, physiology, and secondary metabolism.

Nitrogen uptake by plants is mostly restricted to the uptake of NO₃^– or NH₄⁺ (Gerendás et al., 1997; Hawkesford et al., 2012). Following uptake, the N ions assimilates into amino-acids to build proteins and other N metabolites (Lea and Morot-Gaudry, 2001). As NO₃ needs to be initially reduced to NH₄ in order to be assimilated, and NO₃ uptake and reduction are energy-consuming processes (Crawford, 1995; Ohyama, 2010), NO₃ metabolism is less efficient energetically than NH₄ metabolism (Jones and Jacobsen, 2005; Britto and Kronzucker, 2013). As NO₃^– is an anion, and NH₄⁺ is a cation, the electrochemical mechanisms for their uptake into roots are different and induce different impacts on the plant and its environment. NO₃ may inhibit root uptake and accumulation of other nutrients (Anjana and Iqbal, 2007; White, 2012), and similarly, NH₄ may compete with other cations for root uptake and was demonstrated to inhibit K, Ca, and Mg uptake (Cox and Reisenauer, 1977; Rayar and van Hai, 1977; Bernstein et al., 2005, 2011; White, 2012). Furthermore, the supplied N form may have a direct impact on the uptake, translocation, and accumulation of N in the plant (Britto and Kronzucker, 2002, 2005; Zhang et al., 2019). Another issue associated with N uptake is the different impact of the two N forms on the pH of the rhizosphere. When the root absorbs NH₄, H⁺ is released to the rhizosphere, causing rhizosphere acidification, accompanied by organic acid biosynthesis in the root cells (Britto and Kronzucker, 2005; Neumann and Römheld, 2012). Conversely, NO₃ influx causes rhizosphere alkalinization, accompanied by organic acid biodegradation (Crawford and Glass, 1998; Neumann and Römheld, 2012). Hence, NH₄/NO₃ ratio has a critical influence on the plant energy status, plant and rhizosphere pH adjustment, mineral uptake, and other key metabolic and regulatory processes that determine the plants’ physiological and horticultural performances.

Although N is a macronutrient essential for all plants in high amounts, N oversupply may severely impact plant metabolism and function, and in some cases may be lethal (Britto et al., 2001; Albornoz, 2016) by mechanisms that involve mainly NO₃ and NH₄ uptake and assimilation by the root (Britto and Kronzucker, 2002; Hawkesford et al., 2012; White, 2012). Therefore, an oversupply of NO₃ or NH₄ may be fatal to plants, and the nutritional demands of crops should be studied. No information is available today about the effect of NH₄/NO₃ ratio and NO₃ or NH₄ toxicity on plant function and secondary metabolism in C. sativa.

Plant species may differ in preference for NO₃-based nutrition or NH₄-based nutrition, considering their ability to utilize and absorb NO₃, regulate the rhizosphere pH, and cope with a high supply of NH₄ (Feng and Barker, 1990; Britto and Kronzucker, 2002). As a result of the negative and positive physiological effects of each NO₃ and NH₄, most agricultural plants perform best under a combined NH₄ and NO₃ supply, at the range of 10–30% NH₄/NO₃ ratio (Errebhi and Wilcox, 1990a; Ben-Oliel et al., 2005; González García et al., 2009). Moreover, studies conducted on different plant species indicate that supplying to the plants both NH₄ and NO₃ compared with only one of these N forms, could induce an increase in plant secondary metabolism (Fritz et al., 2006; Sharafzadeh et al., 2011; Saadatian et al., 2014; Zhu et al., 2014; Zhang et al., 2019). Since the response of “drug-type” medical cannabis in particular and C. sativa in general to NO₃ and NH₄ supply are yet unknown, it is difficult to predict the NH₄/NO₃ ratio required for optimal cannabis cultivation.

We have recently demonstrated that in medical “drug-type” cannabis, optimal plant function and development, are achieved under 160 mg L^–1 N at both the vegetative growth phase (Saloner and Bernstein, 2020) and the flowering stage (Saloner and Bernstein, 2020), with a negative correlation between N accumulation and plant secondary metabolism. Cannabinoid and terpenoid production was found to be suppressed by the elevation of N supply, while the plant physiological function and inflorescence yield increase with the elevation of N supply up to 160 mg L^–1 N (Saloner and Bernstein, 2021). Regardless of the recent considerable progress in our understanding of medical cannabis N nutritional requirements, all the available studies were conducted using a uniform ratio of NH₄/NO₃, and the effect of NH₄/NO₃ ratio on medical cannabis remains unknown.

In the present study, we therefore focused on the impact of the ratio between the N sources supplied to the plants (NH₄/NO₃ ratio) on the development, physiology, yield, and secondary metabolism of “drug-type” medical cannabis. The hypothesis guiding the workplan was that NH₄/NO₃ ratio elicits changes to the cannabinoid and terpenoid biosynthesis, as well as affects developmental and physiological characteristics. The project aimed to determine the optimal NH₄/NO₃ ratio for medical cannabis production, and to analyze possible effects of non-optimal ratios. To evaluate the hypothesis, we studied the impacts of five NH₄/NO₃ ratios (0, 10, 30, 50, and 100% N-NH₄, and the remaining N was supplied as N-NO₃), under a uniform N level, on medical cannabis. The results of this study may serve as guidelines for medical cannabis cultivation as part of a pressing need to understand medical cannabis nutritional requirements for a control of yield and secondary metabolites production.

Materials and Methods

Results

Plant Visual Characteristics

The visual appearance of the plant is a primary indicator of the plant physiological state. Plants in the 0–30% NH₄ treatments appeared similar, and demonstrated adequate plant, leaf and inflorescence structure, and color (Figure 1). Nevertheless, the leaves of the 0% NH₄ treatment developed an unfamiliar dotted chlorosis at their margins (Figure 1). The plants of the 50% NH₄ treatment appeared normal throughout most of the experiment duration (Figure 1). However, at the last 2 weeks of the experiment, the leaves became necrotic and wilted, eventually causing death of 40% of the plants in the last week of the experiment. The plants of the 100% NH₄ regime suffered stunted growth and toxicity symptoms already at early stages of the experiment, showing curly leaves with marginal necrosis at the plant growing tips (Figure 2). As the experiment proceeded, the 100% NH₄ plants remained small, and the necrosis spread to all plant parts resulting in plant death (Figure 1). The visual response obtained at the areal parts of the plant is in accord with the development of the roots, which demonstrated optimal development under 0–50% NH₄, and severe damage under 100% NH₄ (Figure 1).

FIGURE 1

Visual appearance of the inflorescences, leaves, whole plants, and roots under increasing N-NH₄ supply. The inflorescence images are of the apical inflorescence on the main stem, and leaf images are of the youngest, fully developed leaf located on the third branch from the plant’s top. Inflorescence, whole plant and root images were taken at maturation (harvest), leaf images were taken 10 days earlier. The brown substance at the base of the roots is a remnant of the coconut fiber plug used for rooting the cuttings at the propagation of the plants.

FIGURE 2

Early symptoms of NH₄ toxicity in medical cannabis. The image was taken 17 days after the initiation of the NH₄/NO₃ fertigation treatments, showing the apical part of a plant receiving a 100% N-NH₄ regime.

Development and Biomass Accumulation

In accord with the visual appearance of the plants, the numerical evaluation as well demonstrated that plant development was significantly influenced by the NH₄/NO₃ ratio. Plant height and inflorescence length decreased with the increase in NH₄ percentage, demonstrating optimal growth under 0–10 and 0% NH₄, respectively, and substantial damage under 100% NH₄ (Figures 3E,F). The inflorescence length exhibited similar response trends to NH₄/NO₃ ratio for the top and side inflorescences, except that side inflorescences were significantly shorter than top inflorescences (Figure 3E). Stem diameter and the number of nodes on the main stem were also smaller in the plants receiving 100% NH₄, and no significant differences were obtained between the other treatments (Supplementary Figure 1).

FIGURE 3

Effect of NH₄/NO₃ ratio on plant biomass and development in medical cannabis plants. Dry weights of inflorescences and whole plant (A), leaves and inflorescence leaves (B), stem and roots (C), % dry weight of inflorescences and whole plant (D), inflorescence length for the top (primary) inflorescence and side (secondary) inflorescence (E), and plant height (F). Presented data are averages ± SE (n = 5). Different small letters above the means represent significant differences between treatments within a curve by Tukey HSD test at α = 0.05.

Plant biomass production generally decreased with the increase in NH₄ supply (Figure 3). Dry biomass of all individual organs, as well as whole plant biomass, were highest under complete NO₃ nutrition (i.e., 0% NH₄), and lowest under 100% NH₄ (Figures 3A–C). Inflorescence biomass, stem biomass, and whole plant biomass were highest under 0% NH₄, significantly lower under 10–50% NH₄, and lowest under 100% NH₄ (Figures 3A,C). Biomass of the fan leaves demonstrated a similar trend, but under 50% NH₄, it was significantly lower than under 10–30% NH₄, while the NH₄/NO₃ regime did not significantly affect biomass of the inflorescence leaves and roots at the 0–50% NH₄ range (Figures 3B,C). Specifically, inflorescence biomass, which may be referred to as the plant yield, decreased by 35, 32, 46, and 97% with the increase of NH₄ supply from 0 to 10, 30, 50, and 100%, respectively (Figure 3A). Since cannabis inflorescences for marketing and consumption are dried to ∼10% moisture-content, economical yields are about 10% higher than the presented inflorescences dry biomasses. The NH₄/NO₃ regime also influenced the percentage of dry weight of the inflorescences and the whole plant: They were significantly higher under 100% NH₄ nutrition compared with the lower NH₄ treatments, amongst no significant difference was obtained (Figure 3D).

Gas Exchange, Water Relations, and Photosynthetic Pigments

The percentage of NH₄ form applied to the medical cannabis plants generated a bi-phasic physiological dose-response (Figure 8). At the concentration range of 0–50% NH₄, most of the physiological parameters examined, including photosynthesis rate, transpiration rate, stomatal conductance, relative water content, osmotic potential, and water use efficiency, were optimal and indicated a proper plant function and a vital physiological state (Figure 8). At the second phase, under the high NH₄ level of 100% NH₄, a severe tissue damage was induced: Plant gas exchange ceased (Figures 8A–D,G), relative water content was very low (Figure 8E), osmotic potential and membrane leakage were very high (Figures 8F,H), and photosynthetic pigment content was close to zero (Figure 9). Excluding the 100% NH₄ treatment, membrane leakage of the leaf tissue demonstrated a unique trend, as the leakage decreased with the elevation of NH₄ percentage, marking a stabilization of low leakage in the range of 30–50% NH₄ (Figure 8H). Contents of photosynthetic pigments in 50% NH₄ plants were significantly lower than of plants receiving lower inputs of NH₄, and significantly higher than of 100% NH₄ plants (Figure 9).

FIGURE 8

Effect of NH₄/NO₃ ratio on gas exchange, membrane leakage, and water relations parameters in medical cannabis plants. Net photosynthesis rate (A), transpiration rate (B), stomatal conductance (C), intercellular CO₂ concentration (D), relative water content (RWC) (E), osmotic potential (F), intrinsic water use efficiency (WUEi) (G), and membrane leakage (H). Presented data are averages ± SE (n = 5). Different small letters above the means represent significant differences between treatments by Tukey HSD test at α = 0.05.

FIGURE 9

Effect of NH₄/NO₃ ratio on the concentration of photosynthetic pigments in medical cannabis plants. Chlorophyll a (A), chlorophyll b (B), and carotenoids (C). Presented data are averages ± SE (n = 5). Different small letters above the means represent significant differences between treatments by Tukey HSD test at α = 0.05.

Discussion

Nitrogen nutrition is one of the major abiotic parameters affecting developmental, physiological, and metabolic processes in plants, including medical cannabis (Saloner and Bernstein, 2020, 2021). As the form of N taken up by the roots affects efficiency and metabolism of N in plants, we aimed to examine the response of medical cannabis to different NH₄/NO₃ ratios. The results reveal a significant sensitivity of medical cannabis plants to NH₄/NO₃ ratios. Plant development, physiological state, inflorescence yield, and secondary metabolite production were all best under NO₃ nutrition when no NH₄ was supplied. Under 10–30% NH₄ supply, the plants did not demonstrate any external or internal toxicity symptoms but produced less yield. At 50% NH₄, plants started to show toxicity injuries, whereas, at 100% NH₄, the plants were substantially damaged by NH₄ toxicity, which was observed already at early stages of the experiment. The results emphasize the significance of nutrition studies in medical cannabis for understanding the plant nutritional requirements and developing an optimal nutritional protocol.

Our results revealed that similar to many other plant species (Goyal and Huffaker, 1984; Wang and Li, 2004; Britto and Kronzucker, 2013; Boschiero et al., 2019), medical cannabis as well functions better under NO₃ nutrition than NH₄ nutrition. The plants which received the low NH₄ levels (0–30% NH₄) demonstrated the best physiological state (Figure 8), with high photosynthetic pigment contents (Figure 9), and consequently were larger and produced higher yield than plants that received higher concentrations of NH₄ (Figure 3). Interestingly, although our latest studies concerning N nutrition in cannabis revealed a correlation between the plant N content and chlorophyll formation (Saloner and Bernstein, 2020, 2021), the data obtained in this study show a negative correlation between the two parameters, implying that high pigment production is better correlated with the plant physiological state than the tissue N content. High NO₃ levels are known to increase rhizosphere pH (Crawford and Glass, 1998; Neumann and Römheld, 2012). In accordance, we obtained a significant increase in leachate pH in response to solely NO₃ nutrition, while ratios of both NO₃ and NH₄ did not alter the pH (Supplementary Figure 2), marking a disadvantage for using NO₃ as the sole N source, especially while growing hydroponically or in an inert media such as perlite.

NH₄ toxicity is a well-documented phenomenon in plants (Goyal and Huffaker, 1984; Britto and Kronzucker, 2002; Roosta and Schjoerring, 2007). In the present study, plants that received only NH₄ suffered from severe NH₄ toxicity, as all developmental and physiological parameters measured were negatively affected, indicating an impaired plant function. The impact was so severe that gas exchange functions of the leaves ceased (Figures 8A–D,G), water content and photosynthetic pigment contents decreased substantially (Figures 3D, ​,8E,8E, ​,9),9), and consequentially the osmotic potential was high and membrane stability was impaired (Figure 8F,H). This response induced a significant decrease in biomass production and development of visual toxicity symptoms (Figures 1–3), leading to plant death. These NH₄ toxicity effects are in accord with previously reported NH₄ toxicity effects in other plant species (reviewed by Britto and Kronzucker, 2002). At the last 2 weeks of the experiment, about 45 days after the initiation of the treatments, plants of the 50% NH₄ treatment as well began to demonstrate toxicity symptoms, resulting in wilting of 40% of the plants in this treatment at the last week of the experiment. As the physiological measurements were conducted before the appearance of the toxicity damage at the last 2 weeks of the experiment, they are not reflected in the physiological data presented in Figure 8, and only the photosynthetic pigment content and the leaves biomass may indicate the initiation of the damage response (Figures 3B, ​,9).9). As was suggested by Britto and Kronzucker (2002), the toxicity damage caused by NH₄ might result from over-time accumulation of NH₄ or other metabolites in the plant tissue. This may explain why the commencement of plant damage occurred earlier under 100% NH₄ than under 50% NH₄ (Figure 2), showing that in C. sativa the NH₄ toxicity damage is cumulative over time. An increase in % NH₄ in the treatments entails an accompanied decrease in NO₃, and vice versa. Therefore, the plant damage obtained under high NH₄ (50–100% NH₄) may reflect also effects of low or lack of NO₃ supply. Similarly, the optimal plant function observed under 0% NH₄ (and 100% NO₃) may result also from the high NO₃ supply, which increased uptake of essential minerals such as Mg and Ca (Figure 7), and enabled proper gas exchange and chlorophyll production (Figures 8, ​,99).

The mechanism for NH₄ toxicity was widely investigated and elaborated on by many studies, and numerous possible negative influences of NH₄ nutrition were considered. These include a decrease in chlorophyll content and hence photosynthetic activity, a reduction in carbohydrate production and the associated decrease in energy availability, increased proton efflux and unstable root pH homeostasis, oxidative stress, futile transmembrane NH₃/NH₄ cycling, increased accumulation of toxic NH₃ and associated tissue damage, and decreased uptake and accumulation of vital cations (Goyal and Huffaker, 1984; Britto and Kronzucker, 2002, 2005; Roosta and Schjoerring, 2007; Neumann and Römheld, 2012; Coskun et al., 2013; Bittsánszky et al., 2015). The results we present in this study demonstrate that the NH₄ toxicity response of cannabis is an integration of the mechanisms mentioned above: (i) Reduced contents of photosynthetic pigments (Figure 9) severely inhibited gas exchange parameters and photosynthesis (Figure 8). (ii) The substantial increase in membrane leakage (Figure 8H) indicates an oxidative damage induced by excess of NH₄ or by a direct toxic effect of overexposure to NH₃. (iii) A decrease in rhizosphere pH (Supplementary Figure 2) may damage root homeostasis, and decrease water uptake and leaf water content. (iv) The substantial decrease in tissue concentration of the essential macronutrients Ca and Mg (Figures 7E,F) likely caused by competition for uptake between NH₄ and these cations, may exert deficiency damages, weakening the plants further.

The effects of NH₄/NO₃ ratio on the cannabis plant ionome that we report in this study are in accord with known cation-anion interactions for plant uptake and in-planta translocation. The results reveal a significant interaction between the N form supplied, and the ability of the cannabis plant to absorb and transport nutrients (Figure 7). First, NO₃ accumulation in the plants increased with the increase in the concentration of NO₃ supplied, demonstrating a relation between supply and accumulation of NO₃. This confirms once again that different NH₄/NO₃ ratios induce changes in N accumulation. This was also apparent from the total N analysis, as total N concentration generally increased with the increase in NH₄ supply, implying that higher NH₄ promoted N uptake and translocation of N to the leaves and inflorescence, and thus has a direct effect on N metabolism. These results for N-NO₃ and total N accumulation are supported by studies of other plant species (Haynes, 1986; Britto and Kronzucker, 2002, 2005; Ohyama, 2010; Zhang et al., 2019). The results show a considerable effect of NH₄/NO₃ ratio on Ca and Mg nutrition. As Ca and Mg are cations which may compete with NH₄, their uptake, translocation and accumulation in the cannabis plants were negatively influenced by the increase in NH₄ supply, and their concentration in the plant organs decreased significantly (Figures 7E,F), as was shown for other plant species (Errebhi and Wilcox, 1990a,b; Bernstein et al., 2005; Roosta and Schjoerring, 2007; Boschiero et al., 2019). Unexpectedly, the increased NH₄ supply resulted in a moderate increase in K concentration in most plant organs (Figure 7C). Nevertheless, the overall amount of K in the entire plant decreased with the increase in NH₄ supply (data not shown), and hence the total plant-level accumulation of K correlates with the well known competition for root uptake usually found between the two cations (Helali et al., 2010; Hawkesford et al., 2012; White, 2012). The concentration of P, which is mainly taken up in the form of the anion H₂PO₄^– (at the experimental pH range), generally decreased with the increase in NO₃^– supply, marking an antagonistic anion-anion relationships between NO₃^– and H₂PO₄^– uptake in cannabis, which was demonstrated for other plant species as well (Kinjo and Pratt, 1971; Feigin, 1990; Serna et al., 1992; Gniazdowska and Rychter, 2000). The concentration of the micronutrients Mn, Zn, Fe, and Cu was only moderately affected by the NH₄/NO₃ ratio, and the most pronounced responses, as well as the highest accumulation levels, were found in the roots (Figures 7G–J). The micronutrient accumulation results, showing highest accumulation in the roots and sensitivity to extreme shortage/toxicity of other minerals, matches the trends we reported previously for micronutrients accumulation in medical cannabis (Saloner et al., 2019; Saloner and Bernstein, 2020, 2021; Shiponi and Bernstein, 2021a).

Conclusion

The response of medical cannabis to different NH₄/NO₃ ratios was analyzed. The highest inflorescence yield and secondary metabolite contents were obtained under sole NO₃ supply. It is therefore proposed that N-NO₃ nutrition is optimal for medical cannabis plant function. Nevertheless, as moderate levels of 10–30% NH₄ (20–60 ppm NH₄) did not induce physiological damage, had only little adverse effect on the inflorescence and cannabinoid yield, and prevented pH changes and leaf tip burns caused by only NO₃ nutrition, it seems safe to suggest to utilize these ratios for medical cannabis cultivation. As NH₄ toxicity damage appeared under 50% NH₄ nutrition (100 ppm NH₄), and severe toxicity symptoms and plant death were obtained under the 100% NH₄ regime, it is not recommended to use levels higher than 30% NH₄ for medical cannabis cultivation. The results suggest that total N concentration in the inflorescence, and not N-NO₃ concentration, is the governing factor affecting cannabinoid and terpenoid biosynthesis.

Similar to other crop species, indications for some genotypic variability in plant response to mineral supply in available for cannabis (Yep et al., 2020; Shiponi and Bernstein, 2021b). This suggest that some fine-tuning may be required by the growers for adjusting N supply and NH₄/NO₃ ratios for specific cultivars.

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Material.

Author Contributions

NB planned the experiments. AS carried out the experiments. Both authors wrote the manuscript, and approved the submitted version.

Conflict of Interest

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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Acknowledgments

References