Genetic diversity and population structure analyses of Plectranthus edulis (Vatke) Agnew collections from diverse agro-ecologies in Ethiopia using newly developed EST-SSRs marker system.

BACKGROUND: Plectranthus edulis (Vatke) Agnew (locally known as Ethiopian dinich or Ethiopian potato) is one of the most economically important edible tuber crops indigenous to Ethiopia. Evaluating the extent of genetic diversity within and among populations is one of the first and most important steps in breeding and conservation measures. Hence, this study was aimed at evaluating the genetic diversity and population structure of this crop using collections from diverse agro-ecologies in Ethiopia. RESULTS: Twenty polymorphic expressed sequence tag based simple sequence repeat (EST-SSRs) markers were developed for P. edulis based on EST sequences of P. barbatus deposited in the GenBank. These markers were used for genetic diversity analyses of 287 individual plants representing 12 populations, and a total of 128 alleles were identified across the entire loci and populations. Different parameters were used to estimate the genetic diversity within populations; and gene diversity index (GD) ranged from 0.31 to 0.39 with overall mean of 0.35. Hierarchical analysis of molecular variance (AMOVA) showed significant but low population differentiation with only 3% of the total variation accounted for variation among populations. Likewise, cluster and STRUCTURE analyses did not group the populations into sharply distinct clusters, which could be attributed to historical and contemporary gene flow and the reproductive biology of the crop. CONCLUSIONS: These newly developed EST-SSR markers are highly polymorphic within P. edulis and hence are valuable genetic tools that can be used to evaluate the extent of genetic diversity and population structure of not only P. edulis but also various other species within the Lamiaceae family. Among the 12 populations studied, populations collected from Wenbera, Awi and Wolaita showed a higher genetic diversity as compared to other populations, and hence these areas can be considered as hot spots for in-situ conservation as well as for identification of genotypes that can be used in breeding programs.

Total biosynthesis of the cyclic AMP booster forskolin from Coleus forskohlii.

Forskolin is a unique structurally complex labdane-type diterpenoid used in the treatment of glaucoma and heart failure based on its activity as a cyclic AMP booster. Commercial production of forskolin relies exclusively on extraction from its only known natural source, the plant Coleus forskohlii, in which forskolin accumulates in the root cork. Here, we report the discovery of five cytochrome P450s and two acetyltransferases which catalyze a cascade of reactions converting the forskolin precursor 13R-manoyl oxide into forskolin and a diverse array of additional labdane-type diterpenoids. A minimal set of three P450s in combination with a single acetyl transferase was identified that catalyzes the conversion of 13R-manoyl oxide into forskolin as demonstrated by transient expression in Nicotiana benthamiana. The entire pathway for forskolin production from glucose encompassing expression of nine genes was stably integrated into Saccharomyces cerevisiae and afforded forskolin titers of 40 mg/L.

Cloning and expression analysis of chalcone synthase gene from Coleus forskohlii.

Flavonoids are an important class of secondary metabolites that play various roles in plants such as mediating defense, floral pigmentation and plant-microbe interaction. Flavonoids are also known to possess antioxidant and antimicrobial activities. Coleus forskohlii (Willd.) Briq. (Lamiaceae) is an important medicinal herb with a diverse metabolic profile, including production of a flavonoid, genkwanin. However, components of the flavonoid pathway have not yet been studied in this plant. Chalcone synthase (CHS) catalyses the first committed step of flavonoid biosynthetic pathway. Full-length cDNA, showing homology with plant CHS gene was isolated from leaves of C. forskohlii and named CfCHS (GenBank accession no. KF643243). Theoretical translation of CfCHS nucleotide sequence shows that it encodes a protein of 391 amino acids with a molecular weight of 42.75 kDa and pI 6.57. Expression analysis of CfCHS in different tissues and elicitor treatments showed that methyl jasmonate (MeJA) strongly induced its expression. Total flavonoids content and antioxidant activity of C. forskohlii also got enhanced in response to MeJA, which correlated with increased CfCHS expression. Induction of CfCHS by MeJA suggest its involvement in production of flavonoids, providing protection from microbes during herbivory or mechanical wounding. Further, our in silico predictions and experimental data suggested that CfCHS may be posttranscriptionally regulated by miR34.

Plant Omics: Isolation, Identification, and Expression Analysis of Cytochrome P450 Gene Sequences from Coleus forskohlii.

The omics analyses of plants and the agrigenomics field offer the opportunity to better characterize our ecosystems. In this context, characterization of cytochrome P450 genes (CYP450s), which constitute one of the largest gene families in plants, is important. They play vital roles in biosynthesis of secondary metabolites, phytohormones as well as in detoxification of harmful chemicals. Tuberous roots of Coleus forskohlii accumulate forskolin, a potent and reversible activator of adenylate cyclase, as well as other related diterpenoids. Coleus forskohlii is also known to produce rosmarinic acid, genkwanin (7-O-methylapigenin), and guaiacol glycerin. We report here the isolation of CYP450s from C. forskohlii, expression profiling of CYP450s in different tissues, and how different elicitors/stresses regulate the expression of different CYP450 sequences. Degenerate primers, designed from the conserved regions of CYP450s, were used to amplify fragments from cDNA of C. forskohlii and a library was prepared. Sequences homologous to CYP450s were assembled into seven distinct gene fragments (CfP450C1-C7), belonging to seven CYP450 families. Expression profiling of CYP450s showed that the transcripts of CfP450C1, CfP450C4, CfP450C5, CfP450C6, and CfP450C7 were prominent in aerial tissues (flower, young leaf, and mature leaf), whereas expression of CfP450C3 was dominant in root and root tip. CfP450C2 showed higher expression in flowers and roots as compared to other tissues. Expression profiles of CYP450s, in response to different stresses (abscisic acid, methyl jasmonate, salicylic acid, 2, 4-dichloro-phenoxyacetic acid, UVA, and wounding) were also studied. This study has isolated CYP450s from C. forskohlii, and will help to understand their regulation as well as their functions. This is the first report on the isolation and expression analysis of CYP450s from this herb.

Metabolic Engineering of Synechocystis sp. PCC 6803 for Production of the Plant Diterpenoid Manoyl Oxide.

Forskolin is a high value diterpenoid with a broad range of pharmaceutical applications, naturally found in root bark of the plant Coleus forskohlii. Because of its complex molecular structure, chemical synthesis of forskolin is not commercially attractive. Hence, the labor and resource intensive extraction and purification from C. forskohlii plants remains the current source of the compound. We have engineered the unicellular cyanobacterium Synechocystis sp. PCC 6803 to produce the forskolin precursor 13R-manoyl oxide (13R-MO), paving the way for light driven biotechnological production of this high value compound. In the course of this work, a new series of integrative vectors for use in Synechocystis was developed and used to create stable lines expressing chromosomally integrated CfTPS2 and CfTPS3, the enzymes responsible for the formation of 13R-MO in C. forskohlii. The engineered strains yielded production titers of up to 0.24 mg g(-1) DCW 13R-MO. To increase the yield, 13R-MO producing strains were further engineered by introduction of selected enzymes from C. forskohlii, improving the titer to 0.45 mg g(-1) DCW. This work forms a basis for further development of production of complex plant diterpenoids in cyanobacteria.

Selection of DNA barcoding loci and phylogenetic study of a medicinal and endemic plant, Plectranthus asirensis J.R.I. Wood from Saudi Arabia.

Genuine medicinal plant materials are very important for potential crude drug production, which can be used to cure many human diseases. DNA barcoding of medicinal plants is an effective way to identify adulterated or contaminated market materials, but it can be quite challenging to generate barcodes and analyze the data to determine discrimination power. The molecular phylogeny of a plant species infers its relationship to other species. We screened the various loci of the nuclear and chloroplast genome for the barcoding of Plectranthus asirensis, an endemic plant of Saudi Arabia. The chloroplast genome loci such as rps16 and rpoB showed maximum similarity to taxa of the same and other genera via BLAST of the National Center for Biotechnology Information (NCBI) GenBank database; hence, they are less preferable for the development of a DNA barcode. However, nrDNA-ITS and chloroplast loci rbcL and rpoC1 showed less similarity via BLAST of the NCBI GenBank database; therefore, they could be used for DNA barcoding for this species.

Authentication of medicinal plant botanical identity by amplified fragmented length polymorphism dominant DNA marker: inferences from the Plectranthus genus.

In Brazil, Plectranthus species are known as "boldo" and have been used in popular medicine for analgesic and dyspeptic purposes. Plectranthus need to be well identified in order to be used as commercially genuine medicinal plants. Here we describe AFLP DNA patterns able to distinguish among different Pectranthus species. The genetic variability of P. grandis Cramer, P. barbatus Andr. and P. ornatus Codd was analyzed with two sets of AFLP primers allowing detection of 241 loci. A total of 22 monomorphic loci were identified in P. barbatus, 15 in P. grandis and 30 in P. ornatus. Among these, 5 loci were informative and species-specific to P. barbatus, 3 to P. grandis and 2 loci were unique to P. ornatus. The AFLP pattern analyzed by different clustering methods assembled individuals according to their species. So far, AFLP represents a genuine and strong method to certify medicinal plant materials.