Response of Medical Cannabis (Cannabis sativa L.) to Nitrogen Supply Under Long Photoperiod

Abstract

Introduction

The Cannabis sativa industry is rapidly evolving worldwide, with an escalating demand for agricultural products for the medicinal and recreational markets (UNODC, 2018; Chouvy, 2019). In spite of the long-time use by humanity for medical, commercial, and recreational purposes, science-based information on the cannabis plant is restricted due to its legal state. Understanding of the plant biology and physiology is needed for supporting the development of modern cultivation schemes.

Phenological stages of cannabis development have been proposed by several authors (Farag and Kayser, 2017; Mishchenko et al., 2017; Raman et al., 2017; Spitzer-Rimon et al., 2019). The development progression includes establishment of the vegetative plant body under long photoperiod, followed by a reproductive development stage under short photoperiod. During growth under long photoperiod, the main stem of the cannabis plants branches monopodially, producing alternate lateral branches. The lateral branches produce second- and third-order branches. Following a transition to short photoperiod, the development of the shoot changes, and the apical buds of the main stem and of primary branches, as well as lateral buds, develop inflorescences that are the basis for commercial yield in drug-type cannabis. Plant architecture at the end of the long photoperiod therefore profoundly affects its reproduction potential and hence yield capacity under short photoperiod. In horticultural practice, cannabis is propagated by rooted cuttings (Cervantes, 2006; Caplan, 2018) and many nurseries are therefore focusing nowadays on growth under long photoperiod, in an attempt to produce high-standard plants.

Mineral nutrition is one of the main factors affecting vegetative development of the plant body (Lea and Morot-Gaudry, 2001; Hawkesford et al., 2012), which dictates the reproductive potential. Understanding the nutritional needs and the physiological and morphological responses of the medical cultivars to mineral nutrition will aid in improving schemes for nutrient supply and plant function. Nitrogen (N) is the most abundant mineral element in plants (Ohyama, 2010). Due to its many roles in plant metabolism, including protein, chlorophyll, and nucleic acid synthesis, N supply is one of the key factors regulating plant growth (Hawkesford et al., 2012). As a result, plant development and metabolism are highly affected by N supply, and N nutrition is one of the prime environmental factors influencing plant function and production (Hauck et al., 1984; Lea and Morot-Gaudry, 2001). Therefore, we chose to examine the response of medical cannabis to N supply in the vegetative growth phase.

Very little information is available about N nutrition of medical cannabis. Available studies into N effects on C. sativa focused mainly on hemp, cultivated for fiber or seed production. Optimal N inputs for fiber growth were reported by numerous studies to range from 60 to 150 kg N ha⁻¹, and higher N levels to increase fiber yield but impair fiber strength (Ehrensing, 1998). Plant biomass, seed yield, and protein contents were reported to increase with N supply up to 120 kg N ha⁻¹ (Vera et al., 2004) or 200 kg N ha⁻¹ (Aubin et al., 2015), and plant growth and inflorescence size increased up to 240 kg N ha⁻¹ supply (Papastylianou et al., 2018). Contradictory results were reported for N effects on secondary metabolism in hemp: N supply (between 0 and 125 ppm) did not affect THC contents in inflorescences (Coffman and Gentner, 1977), while high N supply [above 450 mg N kg(soil)⁻¹] reduced THC contents in leaves (Bócsa et al., 1997). Moreover, N supply was reported to effect sex expression in hemp (Van der Werf and Van den Berg, 1995). It should be considered that hemp has been domesticated and bred for fiber production and hence has different genetic, morphological, and developmental characteristics than the drug-type cannabis that likely entail different nutritional needs. Therefore, it is difficult to derive the drug-type response from information obtained for hemp.

Only a few studies investigated the impact of mineral nutrition in general, and N nutrition in particular, on drug-type cannabis. Recent studies from our group shed some light on various aspects of mineral nutrition influence on medical cannabis development, physiology, and secondary metabolism. We demonstrated an interplay between plant morphology, physiology, and chemistry throughout the plant (Bernstein et al., 2019a), and reported variability along the plant height and between plant organs in cannabinoid contents (including THC and CBD), concentrations of inorganic nutrients, and physiological characteristics (Bernstein et al., 2019a). We showed, for the first time, that nutritional supplements such as humic acids, NPK, and P induce changes in cannabinoid contents in medical cannabis (Bernstein et al., 2019b). In a study that focused on K nutrition, we reported that the response of medical cannabis to K at the vegetative growth phase varied between genotypes, revealing genetic differences within the C. sativa species to mineral nutrition (Saloner et al., 2019); 60 mgL⁻¹ K was the lowest K concentration optimal for growth and metabolism; and 240 ppm was excessive and detrimental for development and function in one genotype, but stimulated rather than restricted development in the other genotype (Saloner et al., 2019). Additionally, under organic fertilization, 389 mgL⁻¹ was the optimal level of N for growth and yield production (Caplan et al., 2017). The extent of N accumulation in the plant was higher in the leaves > roots > stem (Saloner et al., 2019).

The present study focused on N nutrition at the vegetative growth stage. The hypothesis guiding the workplan was that N supply induces morphological and developmental changes in medical cannabis that are associated with changes in the plant ionome, carbon fixation, and tissue water relations. To test this hypothesis, we investigated effects of N inputs ranging from 30 to 320 mgL⁻¹ N in the nutrient solution on physiological and morphological characteristics and mineral accumulation in the plant organs. The information obtained contributes to our understanding of medical-cannabis physiology and mineral nutrition, and directs optimal fertigation regimes for improved production.

Materials and Methods

Results

Discussion

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, ​,5B5B).

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).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, ​,5B5B).

Conclusion

In summary, we found growth retardation under low N supply (30–80 mgL⁻¹) to result from restricted photosynthetic pigments biosynthesis, carbon fixation, and impaired water relations. Excess uptake of N under supply larger than the optimal level of 160 mgL⁻¹ N induced growth retardation by ion-specific toxicity or an indirect induced restriction of carbon fixation. We conclude that the morpho-physiological function under long photoperiod is optimal at 160 mgL⁻¹ N, in the medical cannabis cultivar tested.

Data Availability Statement

All datasets presented in this study are included in the article/supplementary material.

Author Contributions

NB planned the experiments. AS carried out the experiments. NB and AS wrote the manuscript. Both authors contributed to the article 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.

Acknowledgments

Footnotes

References