Gene identification in black cohosh (Actaea racemosa L.): expressed sequence tag profiling and genetic screening yields candidate genes for production of bioactive secondary metabolites

Abstract

Black cohosh (Actaea racemosa L., syn. Cimicifuga racemosa, Nutt., Ranunculaceae) is a popular herb used for relieving menopausal discomforts. A variety of secondary metabolites, including triterpenoids, phenolic dimers, and serotonin derivatives have been associated with its biological activity, but the genes and metabolic pathways as well as the tissue distribution of their production in this plant are unknown. A gene discovery effort was initiated in A. racemosa by partial sequencing of cDNA libraries constructed from young leaf, rhizome, and root tissues. In total, 2,066 expressed sequence tags (ESTs) were assembled into 1,590 unique genes (unigenes). Most of the unigenes were predicted to encode primary metabolism genes, but about 70 were identified as putative secondary metabolism genes. Several of these candidates were analyzed further and full-length cDNA and genomic sequences for a putative 2,3 oxidosqualene cyclase (CAS1) and two BAHD-type acyltransferases (ACT1 and HCT1) were obtained. Homology-based PCR screening for the central gene in plant serotonin biosynthesis, tryptophan decarboxylase (TDC), identified two TDC-related sequences in A. racemosa. CAS1, ACT1, and HCT1 were expressed in most plant tissues, whereas expression of TDC genes was detected only sporadically in immature flower heads and some very young leaf tissues. The cDNA libraries described and assorted genes identified provide initial insight into gene content and diversity in black cohosh, and provide tools and resources for detailed investigations of secondary metabolite genes and enzymes in this important medicinal plant.

Black cohosh: Coming full circle?

“…NMS demonstrates an affinity for serotonin receptors 5-HT1A and 5-HT7, providing a possible mechanism by which black cohosh ameliorates hot flashes (Burdette et al, 2003;Hedlund and Sutcliffe, 2004;Powell et al, 2008). Interestingly, NMS is present in black cohosh in micromolar concentrations and genetic analysis of the plant revealed that expression of genes encoding for enzymes instrumental in serotonin biosynthesis is negligible and inconsistent (Spiering et al, 2011).…”

Efficient, Season-Independent Seed Germination in Black Cohosh (<i>Actaea racemosa</i> L.)

“…The approach appears independent of genotype, and relies on temperature stratification to overcome the low yields of plants due to double dormancy. This method has been used to generate a robust collection of seedlings for transplantation with a high survival rate, and for use as young plant material in studies aimed at developing cell culture, transformation and functional genomics approaches to study gene-metabolite relationships in this important non-model medicinal plant [21].…”

“…The approach provides a basis for undertaking increased cultivation of black cohosh from seed on a year round basis. The approach described for generating young tissues from germinated seedlings has been instrumental in developing a transformation system and plant cell cultures for the application of modern functional genomics approaches to the study of biosynthesis of bioactive secondary metabolites in black cohosh [21].…”

Nitrogen-Containing Constituents of Black Cohosh: Chemistry, Structure Elucidation, and Biological Activities

“…A candidate gene encoding an unspecified amine oxidase has been identified by Spiering et al [53]. N -Guanidinopyrroline may through yet to be identified steps be converted into cimipronidine.…”

“…Another critical molecule in the biosynthetic pathways of isoquinoline alkaloids is reticuline ( 27 ), which was identified at level 1 by comparison with authentic standard. The study by Spiering et al identified a candidate gene for N -methylcoclaurine 3-hydroxylase, an enzyme involved in the biosynthesis of reticuline, providing a mechanistic basis for the origin of this group of alkaloids in black cohosh [53]. …”