Genome-wide identification of cannabinoid biosynthesis genes in non-drug type Cannabis (Cannabis sativa L.) cultivar

Abstract

Background: Cannabis sativa cultivars can be classified as marijuana or hemp, depending on its amount of the psychoactive cannabinoid Δ⁹-tetrahydrocannabinolic acid (THCA). Hemp Cheungsam is a non-drug type Cannabis sativa that is characterized by low THCA content. However, the transcripts and expression profile of cannabinoid biosynthesis pathway genes of hemp Cheungsam have not been investigated.

Methods: RNA-sequencing (RNA-seq) was performed on three different tissue types (flower, leaf, and stem) of hemp Cheungsam to understand their transcriptomes. The expression of cannabinoid biosynthesis pathway genes was further analyzed in each tissue type. Multiple sequence alignment and conserved domain analyses were used to investigate the homologs of cannbinoid biosynthesis genes.

Results: We found that the cannabinoid biosynthesis pathway was mainly expressed in the flowers of hemp Cheungsam, similar to other Cannabis cultivars. However, expression of cannabidiolic acid (CBDA) synthase was much higher than THCA synthase and cannabichromenic acid (CBCA) synthase, suggesting that the transcription profile favors CBDA biosynthesis. Sequence analysis of cannabinoid biosynthesis pathway genes suggested the identity of orthologs in hemp Cheungsam.

Conclusions: Cannabinoid biosynthesis in hemp Cheungsam mostly occurs in the flowers, compared to other plant organs. While CBDA synthase expression is high, THCA and CBCA synthase expression is considerably low, indicating lesser THCA biosynthesis and thus low THCA content. Sequence analysis of key genes (CBDA, THCA, and CBCA synthases) of the cannabinoid biosynthetic pathway indicates that orthologs are present in hemp Cheungsam.

Keywords: Cannabis sativa; Hemp; RNA-seq.

Fig. 1

Phenotype of hemp Cheungsam. A Hemp Cheungsam during the vegetative growth stage. B Top-down view of hemp Cheungsam, showing palmate leaves. C Female flowers developing at the cola at 2 w after flower induction. D Developed female flowers at the cola at 5 w after flower induction. Black (A, B) and white (C, D) scale bars, 1 cm

Fig. 2

Comparison of transcriptomes between different tissue types. A Principal component analysis (PCA) plot of sample triplicates. PC1, principal component 1. PC2, Principal component 2. B Volcano plots for all sample comparisons. FPKM values were compared between two samples. Each volcano plot shows the distribution of fold change and Student’s t-test for all transcripts. Differentially expressed genes (DEGs) were identified with fold change > 2 or fold change < 0.5 and P-value < 0.05. Number of up- and down-regulated DEGs are indicated in each plot

Fig. 3

Identification of tissue-specific genes in hemp Cheungsam flower, leaf, and stem. A Venn diagrams show overlap of DEGs identified from all comparisons. DEGs shared between two comparisons are underlined. B Hierarchical clustering heatmap of shared DEGs, showing expression pattern of tissue-specific genes. Red color represents high expression while blue color represents low expression

Fig. 4

Enriched biological process (BP) GO terms in various plant tissue types. GO terms were selected to have Benjamini–Hochberg adjusted P-value (Benjamini) < 0.05. Top 10 GO terms are shown. Gene clusters correspond to Fig. 3B

Fig. 5

Expression of full-length cannabinoid biosynthetic pathway genes in various hemp Cheungsam plant tissues. Expression levels are represented by both Z-score and FPKM value. Red color indicates high Z-score while blue color indicates low Z-score. Each row represents a gene homolog. The number in each box represents FPKM value. Sum(FPKM) refers to total FPKM from flower, leaf, and stem samples. Flower-specific gene is defined as expression in flowers samples being more than five-fold than leaf or stem samples. AAE, ACYL-ACTIVATING ENZYME. OLS/TKS, OLIVETOL SYNTHASE/TETRAKETIDE SYNTHASE. OAC, OLIVETOLIC ACID CYCLASE. PT, AROMATIC PRENYLTRANSFERASE. CBDAS, CBDA synthase. THCAS, THCA synthase. CBCAS, CBCA synthase. F, flower. L, leaf. S, stem

Fig. 6

AAE orthologs in hemp Cheungsam. A Phylogenetic tree of hemp Cheungsam and GenBank database AAE protein sequences. Solid lines indicate actual phylogenetic distance. Dotted lines are used to align all terminals and do not represent phylogenetic distance. Colored boxes indicate four main groups of AAE identified in the phylogenetic tree. Sum(FPKM) refers to total FPKM from flower, leaf, and stem samples. B Multiple protein sequence alignment of CsAAE1 and CsAAE12 with hemp Cheungsam orthologs. Green bar, AMP-binding domain. Blue boxes highlight C-terminus peroxisome targeting signal type 1 (PTS1; Reumann 2004). Blue P indicates predicted peroxisomal AAE

Fig. 7

OLS/TKS orthologs of hemp Cheungsam. A Multiple protein sequence alignment of CsOLS/CsTKS with hemp Cheungsam orthologs. Purple bar, Chalcone and stilbene synthase N-terminal domain. Pink bar, Chalcone and stilbene synthase C-terminal domain. Green bar, 3-Oxoacyl-[acyl-carrier-protein (ACP)] synthase III domain. Yellow bar, ACP synthase C-terminal domain. Orange bar, FAE1/Type III polyketide synthase-like domain. Based on Taura et al. (2009), CHS catalytic triad residues are highlighted in blue, while residues that may be important for substrate specificity or polyketide length are highlighted in red

Fig. 8

PT orthologs in hemp Cheungsam. A Phylogenetic tree of protein sequences of CsPT1, CsPT4, and hemp Cheungsam PT orthologs. Red and blue boxes indicate close orthologs of CsPT4 and CsPT1, respectively. B Multiple sequence alignment of all sequences in (A). Highlighted sequences are conserved PT motifs: NQxxDxxxD (blue), KDxxDxxGD (orange) (de Bruijn et al. 2020). Green bar, UbiA domain. CsPT1 ortholog, blue circle. CsPT4 ortholog, red circle

Fig. 9

Cannabinoid oxidocyclase orthologs of hemp Cheungsam. A Phylogenetic tree of protein sequences of CsCBCAS, CsCBDAS, and CsTHCAS with orthologs. B Multiple sequence alignment of all sequences in (A). Green bar, FAD-binding domain. Yellow bar, BBE-like domain. Blue circle, CBCAS homolog. Green circle, CBDAS homolog. Red circle, THCAS homolog. Closed circles represent full-length sequences with stop codons. Open circles represent partial sequences