Delineating genetic regulation of cannabinoid biosynthesis during female flower development in Cannabis sativa

Abstract

Cannabinoids are predominantly produced in the glandular trichomes on cannabis female flowers. There is little known on how cannabinoid biosynthesis is regulated during female flower development. We aim to understand the rate-limiting step(s) in the cannabinoid biosynthetic pathway. We investigated the transcript levels of cannabinoid biosynthetic genes together with cannabinoid contents during 7 weeks of female flower development. We demonstrated that the enzymatic steps for producing cannabigerol (CBG), which involve genes GPPS, PT, TKS, and OAC, could rate limit cannabinoid biosynthesis. Our findings further suggest that upregulation of cannabinoid synthases, CBDAS and THCAS in a commercial hemp and medical marijuana variety, respectively, is not critical for cannabinoid biosynthesis. The cannabinoid biosynthetic genes are generally upregulated during flower maturation; increased expression occurs coincident with glandular trichome development and cannabinoid production in the maturing flower. The results also suggest that different cannabis varieties may experience discrete transcriptional regulation of cannabinoid biosynthetic genes. In addition, we showed that methyl jasmonate (MeJA) can potentially increase cannabinoid production. We propose that biweekly applications of 100 μM MeJA starting from flower initiation would be efficacious for promoting cannabinoid biosynthesis. Our findings provide important genetic information for cannabis breeding to generate new varieties with favorable traits.

Keywords: Cannabis sativa; cannabinoid biosynthesis; cannabinoid biosynthetic genes; methyl jasmonate; transcriptional regulation.

FIGURE 1

The remaining CBG amount and gene expression of PT1 and PT4 over the course of 7‐week flower development in CW (a, c) and GG (b, d). Y axis on the left show relative expression of PT1 and PT4. Y axis on the right show the percentage of CBG amount in dry flower samples. Data are presented as means ± SE (n = 4). Positive direction error bars are shown for the line. Significant difference of P < .05 (*) and P < .01 (**) was determined by comparing to week 1 using Student's t test

FIGURE 2

Analysis of CBD and THC accumulation with the expression of cannabinoid synthase genes over the course of 7‐week flower development. (a, b) Superimposition of CBD content with CBDAS expression in CW (a) and GG (b). (c, d) Superimposition of THC content with THCAS expression in CW (c) and GG (d). Data are presented as means ± SE (n = 4). Positive direction error bars are shown for the line. Significant difference of P < .05 (*) and P < .01 (**) was determined by comparing to week 1 using Student's t test

FIGURE 3

Expression of upstream genes in the cannabinoid biosynthetic pathway over the course of 7‐week flower development. (a, c, e) Expression of GPPS, TKS, and OAC in CW. (b, d, f) Expression of GPPS, TKS, and OAC in GG. Data are presented as means ± SE (n = 4). Significant difference of P < .05 (*), P < .01 (**), and P < .001 (***) was determined by comparing to week 1 using Student's t test

FIGURE 4

Graphic representation of correlation matrices demonstrates the correlation plot of cannabinoid biosynthetic genes and metabolites for CW (a) and GG (b). Blue indicates a positive correlation, while red indicates a negative correlation. Size and color of circle represent strength of correlation. Figures were generated using the R corrplot package https://github.com/taiyun/corrplot. (*, P < .05; **, P < .01; ***, P < .001). Percentage data was arcsine transformed prior to statistical analysis

FIGURE 5

THC content in White Tangy Haze flowers over the period of 4 weeks (a–d) following application of MeJA. The box whiskers indicate variability outside the upper and lower quartiles (the upper and lower lines of the box) (n = 3). Different letters indicate statistical significance (P < .05) determined by one‐way ANOVA followed by Fisher's LSD. Percentage data were arcsine transformed prior to statistical analysis. The untransformed data are presented