In Silico Identification of MYB and bHLH Families Reveals Candidate Transcription Factors for Secondary Metabolic Pathways in Cannabis sativa L.

Abstract

Plant secondary metabolic pathways are finely regulated by the activity of transcription factors, among which members of the bHLH and MYB subfamilies play a main role. Cannabis sativa L. is a unique officinal plant species with over 600 synthesized phytochemicals having diverse scale-up industrial and pharmaceutical usage. Despite comprehensive knowledge of cannabinoids’ metabolic pathways, very little is known about their regulation, while the literature on flavonoids’ metabolic pathways is still scarce. In this study, we provide the first genome-wide analysis of bHLH and MYB families in C. sativa reference cultivar CBDRx and identification of candidate coding sequences for these transcription factors. Cannabis sativa bHLHs and MYBs were then classified into functional subfamilies through comparative phylogenetic analysis with A. thaliana transcription factors. Analyses of gene structure and motif distribution confirmed that CsbHLHs and CsMYBs belonging to the same evolutionary clade share common features at both gene and amino acidic level. Candidate regulatory genes for key metabolic pathways leading to flavonoid and cannabinoid synthesis in Cannabis were also retrieved. Furthermore, a candidate gene approach was used to identify structural enzyme-coding genes for flavonoid and cannabinoid synthesis. Taken as a whole, this work represents a valuable resource of candidate genes for further investigation of the C. sativa cannabinoid and flavonoid metabolic pathways for genomic studies and breeding programs.

Analysis of Sequence Variability and Transcriptional Profile of Cannabinoid synthase Genes in Cannabis sativa L. Chemotypes with a Focus on Cannabichromenic acid synthase

“…A number of complete and partially annotated genomic sequences have been published in the latest years [ 6 , 17 , 18 , 19 , 20 ] and information about the transcribed genes in different stages and tissues [ 21 ], as well as on putative regulators of secondary metabolites [ 22 , 23 ] is already known.…”

Cannabinoids and Terpenes: How Production of Photo-Protectants Can Be Manipulated to Enhance Cannabis sativa L. Phytochemistry

“…UV radiation, one of the most effective wavelength ranges that induces cannabinoid biosynthesis (THC, THCV, CBD, and CBG), is perceived by several photoreceptors including UVR8, cryptochromes, and phototropins (Sager et al, 1988;Galvão and Fankhauser, 2015). Few studies have attempted to identify the regulatory elements of PSM biosynthetic pathway in cannabis plants (Marks et al, 2009;Bassolino et al, 2020). Some candidate regulatory genes for both cannabinoid and flavonoid biosynthesis(s) have been pinpointed and regulatory proteins identified; CsMYB77 and CsMYB94 for cannabinoid biosynthesis and CsbHLH112 and CsbHLH113 for flavonoid biosynthesis (Bassolino et al, 2020).…”

“…Few studies have attempted to identify the regulatory elements of PSM biosynthetic pathway in cannabis plants (Marks et al, 2009;Bassolino et al, 2020). Some candidate regulatory genes for both cannabinoid and flavonoid biosynthesis(s) have been pinpointed and regulatory proteins identified; CsMYB77 and CsMYB94 for cannabinoid biosynthesis and CsbHLH112 and CsbHLH113 for flavonoid biosynthesis (Bassolino et al, 2020). Both MYB and bHLH superfamilies play key roles in the regulation of secondary metabolism (Hong, 2016).…”

Selection and validation of reference genes for normalization of qRT-PCR data to study the cannabinoid pathway genes in industrial hemp

“…The increasing popularity of hemp-based phytochemicals has spurred the comprehensive analysis of genome and gene expression studies [ 8 – 11 ]. These analyses are necessary to identify the genes involved in secondary metabolite pathways [ 12 , 13 ] and have helped to discover transcription factors which are the key proteins that positively or negatively control the synthesis of secondary metabolites [ 14 , 15 ]. Accurate gene expression studies such as Northern blotting [ 16 ], ribonuclease protection assay (RPA) [ 17 ], serial analysis of gene expression (SAGE) [ 18 ], and quantitative real-time PCR (qRT-PCR) [ 19 ] are essential to confirm genomic and transcriptomic data.…”