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.