Exogenous application of stress-related signaling molecules affect growth and cannabinoid accumulation in medical cannabis (Cannabis sativa L.)

2.2. Plant treatments

Methyl jasmonate, 95% (Sigma-Aldrich), γ-Aminobutyric acid (Sigma-Aldrich), and Salicylic acid (Duchefa Biochemie) were prepared at 0.1, 1, and 10 mM in distilled water with 0.01% (v/v) Tween 20 (Sigma-Aldrich) and sprayed on plant leaves and inflorescences to the point of runoff. Control plants (mock-treated) were sprayed with a 0.01% (v/v) Tween 20 solution. Treatment groups were separated from each other by polycarbonate screens during phytohormone applications to prevent cross-contamination. All plants were treated once per week during the generative stage (8 applications total) until the week before harvesting.

2.3. Sample collection

Samples of fan leaves, sugar leaves, and perigonal bracts were taken one week before harvesting from 3 plants per treatment, and glandular trichomes (sessile and stalked) were counted by examining sections ranging from 4 to 25 mm² (depending on the source’s sample size) under a Motic SMZ171 stereo microscope. The stalked glandular trichomes density (number of trichomes per mm²) in inflorescences was assessed on the adaxial surface of perigonal bracts, and both adaxial and abaxial surfaces in the sugar leaves. Besides, we counted the number of sessile glandular trichomes on the adaxial surface of fan leaves from the same plants.

Plant height, number of inflorescences, and total fresh weight (FW) were recorded at the harvesting date. Plants were dried at 23 ± 1°C for 14 days until they reached 10-12% of the initial FW. Then, apical inflorescence dry weight (ADW), total inflorescences dry weight (FDW) and leaves dry weight (LDW) were measured. Inflorescence-accompanying leaves (“sugar leaves”) were not trimmed and therefore were counted as inflorescence biomass for the ADW and FDW determination. For the determination of cannabinoids, inflorescences, and leaves from every three plants (treatments), or six plants (control) were pooled to get three samples per treatment.

2.4. Analysis of cannabinoids

Inflorescences and leaves samples were analyzed by near infrared spectroscopy (NIRS) for the determination of the content of cannabinoids. A FOSS NIR DS2500 series (FOSS, Hillerød, Denmark, EU) was used for reflectance measurement of the samples from 400 to 2499.5 nm, every 0.5 nm, equipped with a Si detector for the 400-1100 nm region and a PbS detector for the 1100-2500 nm region. For sample measurement, a circular quartz capsule was employed. Spectral absorbance values were recorded in the reflectance mode as log 1/R, where R is the sample reflectance. ISIscan Nova software version 7.9.1.2 was used for spectra acquisition. Additionally, high performance liquid chromatography coupled to a diode array detector (HPLC-DAD) was employed as reference technique to assure the correct performance of NIRS measurements. An Agilent 1260 Infinity series (Agilent Technologies, Inc, Santa Clara, USA) equipped with a G1329B autosampler and a G1316A DAD was employed. For the chromatographic separation, a Restek Raptor ARC18 (150×2.1 mm, 1.8μm particle size, Restek Corporation, Bellefonte, Pennsylvania, USA) column was used. The separation of the compounds was carried out following an isocratic method at a flow rate of 0.37 mL/min. The mobile phase was composed of 73% B (acetonitrile with 0.1% (v/v) formic acid) and 27% A (milli-Q water with 5 mM ammonium formate and 0.1% formic acid) and the oven temperature was set at 35°C. The injected sample volume was 1 µL. Agilent LC OpenLAB software was employed for data treatment.

For HPLC analysis, approximately 0.5 g aliquots of dry plant material were weighed in 50 mL Falcon tubes and extracted with 5 mL of methanol in an ultrasound bath for 30 min. The supernatant was filtered through 0.22 µm syringe nylon filters and collected in 4 mL glass vials. Prior to HPLC-DAD analysis, proper dilutions of the samples were performed. For NIRS measurements, a circular quartz capsule was filled with plant material previously dried and grounded. Samples were gently pressed with a circular metal weight to homogenize the surface and avoid potential measurement interferences from gaps. The CBDA and THCA contents were calculated as mg of cannabinoid produced per plant, either from inflorescences or the leaf dry biomass.

2.5. RNA extraction, primers used and RT-qPCR

Two weeks before harvesting, floral buds (about 1 g) were collected from three random plants per treatment, 24 h after foliar spraying, and frozen in liquid nitrogen. Total RNA was extracted from 100 mg of frozen tissue using the IQ Easy Plus Plant RNA Extraction Mini Kit (iNtRON Biotechnology), following the manufacturer’s recommended protocol for leaf and seed tissues, followed by phenol-chloroform extractions. The RNA concentration and purity were determined spectrophotometrically in a Nanodrop 1000 (Thermo Fisher Scientific ™, USA), and RNA integrity was checked by visualization in a 1.2% (w/v) agarose gel. RNA samples were stored at -80°C until use.

First-strand cDNA was synthesized from 1μg of total RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems), according to the manufacturer’s instructions. The qPCR was performed in an optical 48-well plate with a Step One Real Time PCR System (Applied Biosystems, Foster City, CA, USA). PCR reactions contained 300-900 nM of each primer ( Supplementary Table 1 ), 1 µl of cDNA template (8.33 ng of input RNA), and 2x Power SYBR^® Green PCR Master Mix (Applied Biosystems) in a final volume of 20 µl. The PCR thermal profile used was an initial step of 95° C/10 min, followed by 40 amplification cycles of 95° C/15 s, 60° C/1 min., and a final dissociation protocol to obtain the melting profiles. Three biological and three technical replicates were assessed for each sample. Relative expression values were expressed as fold-change using the comparative CT method (ΔΔCT Method) (Schmittgen and Livak, 2008). Results are expressed as relative values to floral buds of control plants, and as means ± standard error from three biological replicates.

Specific primer pairs for CsTHCAS, CsCBDAS, CsPT4, and two CsOAC were designed with Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) using reference GeneBank sequences ( Supplementary Table 1 ). Two olivetolic acid cyclase genes (CsAOC-1 and CsAOC-2) and seven prenyltransferase-like genes (LOC115713185, LOC115707809, LOC115713148, LOC115713171, LOC115713205, LOC115713215, and LOC115722991) were identified in the CBDRx reference genome (GenBank GCF_900626175.2), but CsPT4 (LOC115713185) was selected for this study as it was the only one found functional in transgenic yeast (Luo et al., 2019). Amplification of three reference genes, Elongation factor 1 alpha, Serine/threonine-protein phosphatase and Tubulin α -1 (Guo et al., 2018; Deguchi et al., 2021), selected based on their stability in C. sativa, was used as an internal control for normalizing the relative expression data. Primer efficiencies were tested by comparison against a standard curve for each gene.

3.2. Effects on the THCA and CBDA production

The effect of SA, MeJA, and GABA on the main cannabinoids (THCA and CBDA) was analysed in flowers and leaves. Some treatments affected the production of cannabinoids in medical cannabis inflorescences (Figure 3A). The production of CBDA was significantly increased (15.6% more than the mock) in 0.1 mM MeJA treatment. The application of 1 mM MeJA produced a smaller CBDA increase (8.42%) but was not significantly different when compared with the control. In contrast, 10 mM SA, 10 mM MeJA, and 1 mM or 10 mM GABA produced a significant reduction in CBDA yields (23.71%, 22.61%, 19.36%, and 19.8%, respectively). The THCA content was reduced significantly in 0.1 mM SA, 10 mM SA, and 10 mM MeJA treatments (17.93%, 28.87%, and 17.62%, respectively).

Figure 3

CBDA (grey) and THCA (black) produced in inflorescences (A), and leaves (B) by plants subjected to different treatments, expressed in mg per plant. Mean ± standard error is represented. Within each cannabinoid, asterisks indicate significant differences with control plants (one-way ANOVA test, followed by two-tailed Dunnett’s test, **p < 0.05, *p < 0.1).

The exogenous application of the phytohormones SA and MeJA also had interesting effects on the production of cannabinoids in the fan leaves (Figure 3B). We observed a significant and high CBDA accumulation in the leaves of plants treated with 0.1 mM SA and 0.1 mM MeJA, which corresponded to an increase of 57% and 43% regarding control plants, respectively. THCA accumulation was higher in plants treated with 0.1 mM SA and 0.1 mM MeJA than in control plants by 53% and 40%, respectively, although the difference was significant only for the former.

3.3. Relative expression profiles of biosynthetic genes

Changes in the relative expression of the genes involved in the final steps of the cannabinoid biosynthesis pathway (CsAOC-1, CsAOC-2, CsPT4, CsTHCAS, and CsCBDAS) were studied to assess the influence of the treatments on the gene expression. We monitored the changes caused by the application of the lowest (0.1 mM) and highest (10 mM) doses of SA, MeJA, and GABA, chosen because most of the significant changes in the THCA and CBDA production were detected in such treatments (Figure 3A). However, the changes measured in the relative gene expression were not significant regarding control plants (Figure 4).

4.2. Low doses of MeJA increase the cannabinoid yield in inflorescences

Cannabis female inflorescences are the richest plant source of the valuable cannabinoids, produced and stored by the stalked glandular trichomes of the florets and sugar leaves. Thus, floral buds are the preferred plant material for the recreational and medical cannabis industry. As shown in Figure 3A, low doses of MeJA were the only ones producing an increase in the CBDA, reaching up to 15.6% more content than control plants for the 0.1 mM MeJA treatment. In addition, the 1 mM treated plants show an increased CBDA content of 8.42%, but not significantly different from the control plants. The THCA yields also increased on the inflorescences developed in the 0.1-1 mM MeJA treated plants by 11% and 5%, respectively, although these differences were not significantly different from the control plants. Therefore, low doses of MeJA, particularly 0.1 mM, are effective in increasing the cannabinoid yield in the inflorescences of medical cannabis. A recent study (Apicella et al., 2022) also reported an increase of the THC content in inflorescences of a marijuana variety (“White Tangy Haze”) sprayed with 0.1-1 mM MeJA, suggesting that this treatment can be species-wide used to enhance cannabinoid yields in Cannabis sativa. On the other hand, our results using SA and GABA contrast with those obtained by Jalali et al. (2019), indicating either that the elicitation effects of exogenously delivered signaling molecules could be affected by the lack of genetic uniformity in the seed-derived population, environmental conditions, or that they could be genotype-dependent to some extent (Schwabe and McGlaughlin, 2019; Toth et al., 2020; Johnson and Wallace, 2021).

4.3. Low doses of SA and MeJA increase the cannabinoid yield in leaves

The cannabis leaves are a minor source of cannabinoids compared with the plant inflorescences. However, leaf biomass represents a significant portion of the whole plant (around 30% of TDW), containing relatively high quantities of cannabinoids in medical cannabis varieties, and is potentially worth as a source for the obtention of extracts and purified compounds for the pharmaceutical industry. Leaves collected from 0.1 mM SA and 0.1 mM MeJA treated plants increased significantly their CBDA yield by 57.31% and 43.18% to control plants, respectively. Also, the THCA yield was increased by as much as 52.68% in the 0.1 mM SA treatment (significant), and by 40.27% in the 0.1 mM MeJA treatment (non-significant) (Figure 3B). Interestingly, this result invites speculation about if the cannabinoid biosynthesis in leaves could be regulated differently than in inflorescences. Nevertheless, both the 0.1 mM SA and MeJA treatments produced a sharp increase in the cannabinoids yield in medical cannabis leaves, improving the profitability of this plant material as a source of cannabinoids.

We want to thank the members of our departments of Plant Cultivation and Breeding, and Analytical Chemistry for their technical participation in this project. We are also grateful to Dra. Eva Madrid and Antonio M. Trasierras for their valuable contributions to this manuscript.