Early floral cannabinoid production

To determine the spatial- and temporal-specific cannabinoid production, the amounts of CBGA, CBG, CBDA, CBD, THCA, THC, and CBN in the immature buds, leaves, and stem tissues were quantified and compared. A previous study reported that the concentration of most cannabinoids in female flowers are two-fold higher than the concentration in the inflorescence leaves (Bernstein et al. 2019b). In this study, flower tissue showed the highest cannabinoid production among three tissues. In flowers, the production of CBDA and THCA appeared to be the most predominant, producing 11,848 μg/g (1.1% w/w) and 12,028 μg/g (1.2% w/w), respectively (Supplementary Fig. 1). The floral production of CBDA were 2.4-fold higher than the production in leaf tissues (n = 24, p < 0.0001) and 25-fold higher than the production in stem tissues (n = 24, p < 0.0001) (Supplementary Fig. 1). Similarly, the production of THCA was 2.3-fold higher production than the production in leaf tissues (n = 24, p < 0.0001) and 29.8% higher than the production in stem tissues (n = 24, p < 0.0001) (Supplementary Fig. 1). Other cannabinoids including CBGA, CBG, CBD, and THC were produced less than 0.05%. However, the levels were consistently high in flower tissues than leaf and stem tissues (Supplementary Fig. 1).

Floral cannabinoid production on day 7, 12, and 14 after the transition to the short-day were measured using HPLC. The cannabinoid analyses showed that the amount of CBDA increased 1.5-fold from 11,848 μg/g (1.1% w/w) at day 7 to 18,204 μg/g (1.8% w/w) at day 14 (Supplementary Fig. 2C). The level of total CBD increased 1.6-fold from 11,090 μg/g (1.1% w/w) at day 7 to 17,705 μg/g (1.7% w/w) at day 14 (Supplementary Fig. 2G). Additionally, the levels of CBD and THC increased 2.5-fold (699 μg/g; 0.06% w/w to 1740 μg/g; 0.1% w/w) and 4-fold (1,089 μg/g; 0.1% w/w to 4320 μg/g; 0.4% w/w), respectively (Supplementary Fig. 2D, F). However, other cannabinoids such as CBGA, CBG, THCA, and total THC remained unchanged during the first 2 weeks of flowering (Supplementary Fig. 2A, B, E, and H).

Leaf cannabinoids were also measured at the same time regime. The production of CBDA and THCA were 4868 μg/g (0.48% w/w) and 5220 μg/g (0.52% w/w), respectively at day 7. However, the concentrations did not change during the early flowering stage (Supplementary Fig. 2A–F).

These results suggest selective activation of flower cannabinoids biosynthetic genes. By deactivating the cannabinoid production in other tissues, presumably the catalytic energy and carbons used in leaf tissues were redirected to the flower organ for concentrating cannabinoid production in the specialized trichome cells. Supplementary Table 1 presents the measured floral cannabinoid concentrations of the immature flower tissues from control plants (day 1) and stress-treated plant (day 6 or day 8) that were used to calculate the production values during that timeframe.

These results are consistent with the previous studies that most cannabinoids production is organ and location–specific, with the trichome-dense flowers being the organs that produce the greatest amount of cannabinoids (Bernstein et al. 2019b). Another study also showed that cannabinoid production can vary greatly among different inflorescence locations with greater concentrations toward the apical meristem (Namdar et al. 2018).

Cannabinoid productions in response to mechanical wounds

Field-grown Cannabis is constantly exposed to adverse biotic (e.g., microbial and herbivore pests), abiotic (e.g., excess heat, drought, and wind), and man-made stresses (e.g., tractor). These stresses have been reported to significantly impact cannabinoid production.

To investigate how mechanical damage affects cannabinoid metabolism, hemp clones (7 weeks old) were tested in grow tents located in a greenhouse. Treatments were started during the first week of flower to elucidate the greatest recordable amount of response, and to avoid the senescence phase of growth. Plants were harvested for the temporal- and spatial-specific cannabinoid analyses 14 days after the transition to the short-day, which limited this study to understanding the effects of these stresses on the onset of flowering; however, future studies should investigate the impact these stresses have on mature flower.

Figure 1 shows the changes of cannabinoid accumulation in three Cannabis tissues—immature flower, leaf, and stem—in response to mechanical wounding. The seven cannabinoid compounds described above were quantified using HPLC. The cannabinoid level observed after 5 days of treatment was compared to the 5-day chemical profile difference shown in the control plants. In the control plants, the cannabinoid production remained about the same level for 5 days. The mechanical wounding also did not impact the level of any cannabinoids (n = 3, p > 0.05) in any tissues of the plants (Fig. 1 and Table 1). In leaf and stem tissues, only CBDA and THCA were quantifiable and appeared not to be changed after mechanical wounding (n = 3, p > 0.05). Other cannabinoid compounds such as CBGA and CBG were under detectable limit in both treated and control plants.

Fig. 1
figure1

No impact on the production of cannabinoids in response to 5 days of short-term mechanical wounding during the first week of flowering. Quantitative comparisons of cannabigerolic acid (A), cannabidiolic acid (B), Δ-tetrahydrocannabinolic acid (C), cannabigerol (D), cannabidiol (E), and Δ-tetrahydrocannabinol (F) production in hemp flower, leaf, and stem tissues in response to 5 days of mechanical wound treatment. The values provided represent the difference between the cannabinoid concentration on day 1 and day 6 of flower. Negative values indicate that the day 6 concentration was lower than the concentration on day 1, whereas a positive value indicates an increase of that cannabinoid over the 6-day period. Statistical analysis was performed using one-way ANOVA, followed by Tukey’s multiple comparisons test. The bar graph represents mean ± s.d. (n = 3)

Table 1 Summary of cannabinoid production per treatment

Several studies have revealed that short-term mechanical damage affects the production of secondary metabolites in cotton (Park et al. 2019b), Catharanthus roseus (Chen et al. 2018), sugar beet (Lafta and Fugate 2011), and lettuce (Saltveit 2000). These studies showed that the production of secondary metabolites (i.e., terpenoids, alkaloids, and phenolic compounds) that play an important role in direct defense were increased by the abiotic stress. Unlike these crops, short-term mechanical stress (1–5 days) did not affect the cannabinoid production in the studied hemp variety. While we did not study long-term effects of this mechanical damage, it appears that 5 days was not sufficient to induce any biosynthetic changes in the phytocannabinoid production. Future studies will be needed to understand the long-term effects on mature flower chemometrics; however, it does not appear to affect a hemps plant ability to produce cannabinoids at the onset of flower.

Cannabinoid productions in response to herbivore stress

Insect pests can be classified by how they infest Cannabis, such as insects with “chewing mouth parts” affect the roots, leaves, stems, and flowers, and “piercing-sucking” insects can bypass insecticidal cannabinoids on the surface of the plant to access the sap within the plant (McPartland 1996). The vast nature of the types of insects that affect Cannabis cultivation provides numerous outcomes when infested and activates various stress responses depending on the insect. Thus, this investigation of the cannabinoid content response to particular insects is essential to field-grown hemp since cannabinoids are one of the most valued products of the Cannabis plant.

To examine how the insect herbivores affect the cannabinoid production and composition, 20 3rd instar caterpillar larvae of tobacco hornworm Manduca sexta were placed on the 7-week-old hemp plants’ leaves for 5 days. It was observed that the larval insects preferentially feed on leaf tissues over stems, flowers, bracts, and petals. Figure 2 shows quantitative comparisons of 5 days of cannabinoid production between control and insect damaged hemp plants. The herbivore wounding significantly reduced the cannabinoid production of CBGA, with the control plants accumulating 308 μg/g (0.03% w/w) to the treated plants losing 24 μg/g (0.0024% w/w) (p < 0.01), CBG, accumulating 69 μg/g (0.0069% w/w) in control plants to losing 52 μg/g (0.0052% w/w) in treated plants (p < 0.05), and CBD, accumulating 755 μg/g (0.075% w/w) in control plants to accumulating 194 μg/g (0.019% w/w) in treated plants (p < 0.05) while other cannabinoids, CBDA, THCA, and THC levels (Fig. 2 and Table 1) remained unchanged during the 7-day observation window. While the reduction of CBGA, CBG and CBD are significant, the treatment procedure could be improved by replacing the porous screen with a non-light blocking insect containment; however, due to the minimal light interference (4.3% photosynthetically active radiation reduction) observed here, it suggests the changes that occurred were due solely to the insect pressure, although future studies should verify these results with differing insect contaminant approaches.

Fig. 2
figure2

Differential cannabinoid production in response to 5 days of short-term herbivore stress during the first week of flowering. Quantitative comparisons of cannabigerolic acid (A), cannabidiolic acid (B), Δ-tetrahydrocannabinolic acid (C), cannabigerol (D), cannabidiol (E), and Δ-tetrahydrocannabinol (F) production in hemp flower, leaf, and stem tissues in response to 5 days of caterpillar larvae M. sexta foraging. The values above indicate the difference in concentrations between day 1 and day 6 of flowering. A negative value indicates a lower concentration after 6 days of flowering than at initiation, whereas a positive value indicates an increase in concentration over that 6-day period. Statistical analysis was performed using one-way ANOVA, followed by Tukey’s multiple comparisons test. The bar graph represents mean ± s.d. (n = 3). **p < 0.01 and *p < 0.05

Manduca sexta caterpillar regurgitates onto the wounded site of a host plant while feeding (Paudel Timilsena and Mikó 2017). Insect regurgitant contains small molecular elicitors such as fatty acid conjugates, inceptin, and calliferins, as well as larger enzymatic molecules such as a glucose oxidase protein (Paudel Timilsena and Mikó 2017). The regurgitant is used to suppress plant defense mechanisms, leading to the changes in metabolic profiles that involve the release of toxic chemicals such as alkaloids, anthocyanins, phenols, and quinones, as well as volatile terpenoids that repel natural enemy insects (War et al. 2012). However, it was unexpected that the insect damage decreased cannabinoid production as they have been previously considered an effective stimulator to stress response (Benelli et al. 2018; Park et al. 2019a). The observed decrease is likely due to cannabinoid biosynthesis not being triggered by this particular herbivore’s stressors, the regurgitant suppressing the plant’s defense response, or the signal to increase production may not have been observable within the limited 5-day window of this study.

Cannabinoid production in response to excess heat

Hemp grows ideally at 24–30 °C in nitrogen-enriched fertilized soils (pH 6.0–7.5) under a regime of 16–24 h of light and 0–8 h of darkness with 40–60% humidity level (Adesina et al. 2020; Chandra et al. 2017). In fields, higher temperatures (> 31 °C) are a common stressor that alters plant physiology and metabolism. To examine how excess heat affects cannabinoid production, six hemp clones were heat-treated at 45–50 °C over 7 days and six clones served as controls at 22–27 °C. Figure 3 shows quantitative comparisons of cannabinoid production between control and heat-treated hemp plants over the 7-day period. Within 3 days, the hemp plants were completely wilted under excess heat regardless of the water supply ( 1L/day). It should be noted that plants were also exposed to water stress in addition to the heat stress due to the increased transpiration caused by the increased temperature. The HPLC demonstrated that excess heat caused significant cannabinoid metabolic changes. In the untreated control plants, female inflorescence produced 206 μg/g (0.02% w/w) of CBGA and 21 μg/g (0.0021% w/w) of CBG during the 7-day observation window. After 7 days of excessive heat treatment, the concentration of CBGA and CBG decreased by 182 μg/g (0.0182% w/w) (n = 3, p < 0.001 and p < 0.05) and by 112 μg/g (0.0112% w/w) (n = 3, p < 0.05), respectively (Fig. 3 and Table 1). Contrastingly, CBDA, THCA, CBD, and THC bioaccumulations were not changed during this 7-day window (Fig. 3 and Table 1).

Fig. 3
figure3

Differential cannabinoids production in response to 7 days of short-term heat stress during the first 2 weeks of flowering. Quantitative comparisons of cannabigerolic acid (A), cannabidiolic acid (B), Δ-tetrahydrocannabinolic acid (C), cannabigerol (D), cannabidiol (E), and Δ-tetrahydrocannabinol (F) production in hemp flower, leaf, and stem tissues in response to 7 days of heat (45–50 oC) stress. The values above indicate the difference in concentrations between day 1 and day 8 of flowering. A negative value indicates a lower concentration after 8 days of flowering than at initiation, whereas a positive value indicates an increase in concentration over that 8-day period. Statistical analysis was performed using one-way ANOVA, followed by Tukey’s multiple comparisons test. The bar graph represents mean ± s.d. (n = 3). ***p < 0.001 and *p < 0.05

As with all cannabinoids, it was expected to see an increase in concentration over the 7-day period; however, heat stress caused the opposite effect. With the decreased concentrations of CBGA and CBG, it would be hypothesized that the enzymatic conversion of CBGA to THCA and CBDA would be happening at a faster rate in the treated plants causing an increase of the terminal cannabinoid concentrations while depleting the precursor pool. However, no significant difference was observed in the concentrations of THCA and CBDA, thus it appears the enzymatic conversion was not affected and the biosynthesis of CBGA may have been downregulated or inhibited upstream in the cannabinoid biosynthetic pathway. While we cannot definitively say whether it was the heat stress or watering habits that affected this cannabinoid production, this result does provide support for a potential downregulation of CBGA production during the initiation of flowering, which would be hypothesized to decrease overall cannabinoid production if the study would have observed longer into the flowering stage.

Cannabinoid productions in response to drought

Figure 4 and Table 1 show the cannabinoid production in response to 7 days of drought stress in the various hemp tissues. Notably, the drought treatment has changed the biosynthesis of CBG, CBD, and THC. Unlike other stresses investigated, drought significantly increased CBG production by 40% while CBD and THC were dramatically reduced by 70–80%. The control plants accumulated 336 μg/g (0.03% w/w) of CBG during the 8-day window, whereas, the amount accumulated in the treated plants significantly increased to 622 μg/g (0.06% w/w) (n = 3, p < 0.001). In addition, two other downstream products, CBD and THC concentrations were significantly decreased from accumulating 1182 μg/g (0.12% w/w) of CBD in the control plants to only accumulating 296 μg/g (0.02% w/w) of CBD in the plants subjected to drought stress (n = 3, p < 0.0001). Similarly, the control plants produced 3927 μg/g (0.39% w/w) of THC during this 8-day period, whereas the plants subjected to drought only accumulated 580 μg/g (0.05% w/w) of THC (n = 3, p < 0.0001).

Fig. 4
figure4

Differential cannabinoids production in response to 7 days of short-term drought stress during the first 2 weeks of flowering. Quantitative comparisons of cannabigerolic acid (A), cannabidiolic acid (B), Δ-tetrahydrocannabinolic acid (C), cannabigerol (D), cannabidiol (E), and Δ-tetrahydrocannabinol (F) production in hemp flower, leaf, and stem tissues in response to 7 days of drought stress. The values above indicate the difference in concentrations between day 1 and day 8 of flowering. A negative value indicates a lower concentration after 8 days of flowering than at initiation, whereas a positive value indicates an increase in concentration over that 8-day period. Statistical analysis was performed using one-way ANOVA, followed by Tukey’s multiple comparisons test. The bar graph represents mean ± s.d. (n = 3). ****p < 0.0001 and ***p < 0.001

CBG is a shared precursor molecule to the production of both CBD and THC. The increase of CBG accumulation might indicate the blockage of conversion into the two downstream intermediates, CBDA and THCA, resulting in accumulation of CBGA that was decarboxylated to CBG under these stress conditions. The catalytic enzymes, CBDA and THCA synthases may have malfunctioned enzymatically and/or their genes’ expression was downregulated resulting in less enzyme available. The reduced enzymatic activity consequently resulted in the decrease of downstream end-products (CBD and THC) while the precursor CBG levels accumulate. Water deficit is directly related to a variety of cellular processes including carbohydrate transport and metabolism, signal transduction mechanisms, and secondary metabolite biosynthesis, transport, and catabolism (Gao et al. 2018). Similar to their findings, in this study, drought stress resulted in the decreased levels of end-product cannabinoids.

Additional work should investigate if drought also affects other secondary metabolites such as terpenoids by measuring terpenoid content in response to drought stress, as well as time-specific drought stresses throughout the flower stage to determine the overall effects on cannabinoid concentration.

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