Secondary metabolite contents and antimicrobial activity of leaf extracts reveal genetic variability of Vernonia amygdalina and Vernonia calvoana morphotypes.

Vernonia species (Asteraceae) are indigenous medicinal and food leaf vegetables commonly consumed in some African countries like Cameroon where they constitute a staple dish called "ndolé." Previous studies have demonstrated the nutritional importance of Vernonia, but there is little knowledge of their agronomic value and genetic potentials. Wide genetic variability in the genus has been established earlier through the study of its pollen. However, to the best of our understanding, no such study has been undertaken on the genetic variability of Vernonia with respect to its secondary metabolites. This study was therefore aimed at evaluating the genetic variability of Vernonia amygdalina (VAA and VALF) and V. calvoana (VCAB, VCAV, VCSB, and VCSV) morphotypes in 2016 based on secondary metabolite content and antimicrobial properties. This involved phytochemical analysis by HPLC/MS for the detection of alkaloids, flavonoids, phenols, and tannins in leaves from each of the six genotypes. Results showed that all tested genotypes are rich in phenols (18 analyzed), flavonoids, and tannins with, VAA richer in phenols (206.1 ± 3.12 µg/g of dry extract), followed by VCAV (197.9 ± 18.03 µg/g). The lowest level of flavonoid was found in VCSV (81.6 ± 7.21 µg/g), while the highest was from VCAB (132.8 ± 31.5 µg/g). VCSV (56.3 ± 4.08 µg/g) had the lowest level of tannins, while VCAB (97.8 ± 23.8 µg/g) had the highest levels. Assessment of antimicrobial activity of leaf extracts from the six genotypes was done by culture on Mueller Hinton (MH) agar and MH broth agar for bacteria and in Sabouraud dextrose agar and Sabouraud dextrose broth media for fungi, respectively. All morphotypes exhibited inhibitory activity on bacteria except VAA, with isoquercetin characteristic of VCSV. Chemotaxonomic analyses of Vernonia morphotypes highlighted the genetic diversity within species and variability of antimicrobial properties of ethanolic leaf extracts among morphotypes. These results provide baseline data in the valuation of genetic resources and the establishment of improvement programs.

Generation and Characterization of a Soybean Line with a Vernonia galamensis Diacylglycerol Acyltransferase-1 Gene and a myo-Inositol 1-Phosphate Synthase Knockout Mutation.

Soybean (Glycine max) meal is an important protein source. Soybean meal with lower phytate and oligosaccharides improves meal quality. A single recessive mutation in soybean myo-inositol 1-phosphate synthase (Gm-lpa-TW75-1) confers a seed phenotype with low phytate and increased inorganic phosphate. The mutant was crossed with high oil lines expressing a diacylglycerol acyltransferase1 (DGAT) gene from Vernonia galamensis (VgD). Gm-lpa-TW75-1 X VgD, designated GV, has 21%, and 22% oil and 41% and 43% protein from field and greenhouse seed production, respectively. No significant differences were found in mineral concentrations except for Fe which was 229 µg/g dry mass for GV followed by 174.3 for VgD and 162 for Gm-lpa-TW75-1. Phosphate (Pi) is higher in Gm-lpa-TW75-1 as expected at 5 mg/g, followed by GV at 1.6 mg/g whereas Jack, VgD, and Taiwan75 have about 0.3 mg/g. The Gm-lpa-TW75-1 line has the lowest phytate concentration at 1.4 mg/g followed by GV with 1.8 mg/g compared to Taiwan75, VgD, and Jack with 2.5 mg/g. This work describes a high oil and protein soybean line, GV, with increased Pi and lower phytate which will increase the nutritional value for human and animal feed.

[Seed-specific expression of heterologous gene DGAT1 increase soybean seed oil content and nutritional quality].

Enhancing soybean (Glycine max) oil production is crucial to meet the market demand of vegetable oil. Diacylglycerol acyltransferase (DGAT) catalyzes the final acylation reaction of triacylglycerol (TAG) synthesis, acting as one of the rate-limiting enzymes for oil biosynthesis in plant seeds. Here, a cDNA clone VgDGAT1A encoding the DGAT1 protein was isolated from the high oil plant Vernonia galamensis. VgDGAT1A was specifically overexpressed in soybean seeds, and several high-generation transgenic lines (T7) were obtained by continuous selection. qPCR analysis showed that VgDGAT1A was highly expressed in the mid-development stage (30-45 DAF) of the transgenic seeds. Accordingly, the DGAT enzyme activity in the transgenic seeds was increased by 7.8 folds in comparison with the wild-type controls. Seed oil and starch contents were, respectively, increased by 5.1% (Dry weight) and reduced by 2%-3% in the transgenic soybeans. Importantly, protein content was not significantly different between transgenic and control seeds. Seed weight and germination rate of the transgenic lines exhibited no negative effect. Fatty acid profiling demonstrated that antioxidant oleic acid (C18:1Δ9) content in the transgenic seed oil was elevated by 8.2% compared to the control, and correspondingly, easily-oxidized linoleic acid (C18:2Δ9,12) and linolenic acid (C18:3Δ9,12,15) were decreased by 6% and 2% respectively. Taken together, seed-specific overexpression of an exogenous VgDGAT1A gene can break the negative linkage of oil and protein contents in soybean seeds, indicating that engineering of this highly-active DGAT enzyme is an effective strategy to improve oil yield and nutritional value in oilseeds.

A Vernonia Diacylglycerol Acyltransferase Can Increase Renewable Oil Production.

Increasing the production of plant oils such as soybean oil as a renewable resource for food and fuel is valuable. Successful breeding for higher oil levels in soybean, however, usually results in reduced protein, a second valuable seed component. This study shows that by manipulating a highly active acyl-CoA:diacylglycerol acyltransferase (DGAT) the hydrocarbon flux to oil in oilseeds can be increased without reducing the protein component. Compared to other plant DGATs, a DGAT from Vernonia galamensis (VgDGAT1A) produces much higher oil synthesis and accumulation activity in yeast, insect cells, and soybean. Soybean lines expressing VgDGAT1A show a 4% increase in oil content without reductions in seed protein contents or yield per unit land area. Incorporation of this trait into 50% of soybeans worldwide could result in an increase of 850 million kg oil/year without new land use or inputs and be worth ∼U.S.$1 billion/year at 2012 production and market prices.

Karyotype characterization of two populations of Vernonia geminata (Asteraceae, Vernonieae) using banding and FISH techniques.

In order to extend our knowledge concerning karyotypes of the genus Vernonia, we applied various techniques of chromosome banding, including AgNOR and triple staining with the fluorochromes CMA/DA/DAPI (CDD), and of fluorescent in situ hybridization (FISH) for the 45S rDNA probe to specimens of two populations of Vernonia geminata collected from an open-pasture area, in southern Brazil. B chromosomes were observed in one of the populations. Both populations of V. geminata presented a pair of CMA(3)(+) terminal bands and one pair of chromosomes with terminal AgNOR banding. The FISH evidenced, in one population, two pairs of small sites of 45S rDNA; these being two small terminal sites and two centromeric sites. In the other population, there was only one pair of small terminal sites and two sites in two B chromosomes, one in each chromosome. There was coincidence of localization between CMA(+) and NOR bands with one of the small terminal sites of 45S rDNA of one chromosome of the normal complement, but not in B chromosomes.

Vernonia DGATs can complement the disrupted oil and protein metabolism in epoxygenase-expressing soybean seeds.

Plant oils can be useful chemical feedstocks such as a source of epoxy fatty acids. High seed-specific expression of a Stokesia laevis epoxygenase (SlEPX) in soybeans only results in 3-7% epoxide levels. SlEPX-transgenic soybean seeds also exhibited other phenotypic alterations, such as altered seed fatty acid profiles, reduced oil accumulation, and variable protein levels. SlEPX-transgenic seeds showed a 2-5% reduction in total oil content and protein levels of 30.9-51.4%. To address these pleiotrophic effects of SlEPX expression on other traits, transgenic soybeans were developed to co-express SlEPX and DGAT (diacylglycerol acyltransferase) genes (VgDGAT1 & 2) isolated from Vernonia galamensis, a high accumulator of epoxy fatty acids. These side effects of SlEPX expression were largely overcome in the DGAT co-expressing soybeans. Total oil and protein contents were restored to the levels in non-transgenic soybeans, indicating that both VgDGAT1 and VgDGAT2 could complement the disrupted phenotypes caused by over-expression of an epoxygenase in soybean seeds.

Increased expression of fructan 1-exohydrolase in rhizophores of Vernonia herbacea during sprouting and exposure to low temperature.

Rhizophores of Vernonia herbacea, an Asteraceae found in the Brazilian Cerrado, store high amounts of fructans that vary in composition over the phenological cycle. Fructan 1-exohydrolase (1-FEH) activity is detectable during the sprouting phase, mainly in the proximal regions of rhizophores, of plants induced to sprout by defoliation and/or cold storage. We found an increase in 1-FEH gene expression during natural and induced sprouting and further enhancement through low-temperature treatment. Furthermore, a comparative analysis of 1-FEH gene expression in different regions of the rhizophores during the transition from dormancy to sprouting is presented. Transcripts were detected mainly in the proximal region, coinciding with high 1-FEH activity and a high concentration of free fructose. Low temperature promoted the accumulation of fructans of a low degree of polymerization (DP) and enhanced 1-FEH activity and gene expression. It is hypothesized that a set of 1-FEH proteins acts in two different ways during fructan mobilization: (1) by hydrolyzing fructo-oligosaccharides and -polysaccharides in sprouting plants (naturally or induced) for carbon supply and (2) by hydrolyzing preferably fructo-polysaccharides under low temperature to maintain the oligosaccharide pool for plant cold acclimation.

Vernonia DGATs increase accumulation of epoxy fatty acids in oil.

Vernolic acid (cis-12-epoxy-octadeca-cis-9-enoic acid) is valuable as a renewable chemical feedstock. This fatty acid can accumulate to high levels in the seed oil of some plant species such as Vernonia galamensis and Stokesia laevis which are unsuitable for large-scale production. A cost-effective alternative for production of epoxy fatty acids is to genetically engineer its biosynthesis in commercial oilseeds. An epoxygenase cDNA (SlEPX) responsible for vernolic acid synthesis and two acyl-CoA : diacylglycerol acyltransferase cDNAs (VgDGAT1 and VgDGAT2) catalysing triacylglycerol (TAG) formation were cloned from developing seeds of S. laevis and V. galamensis. Co-expression of SlEPX and VgDGAT1 or VgDGAT2 greatly increases accumulation of vernolic acid both in petunia leaves and soybean somatic embryos. Seed-specific expression of VgDGAT1 and VgDGAT2 in SlEPX mature soybean seeds results in vernolic acid levels of approximately 15% and 26%. Both DGAT1 and DGAT2 increase epoxy fatty acid accumulation with DGAT2 having much greater impact.

Cloning, characterization and functional analysis of a 1-FEH cDNA from Vernonia herbacea (Vell.) Rusby.

Variations in the inulin contents have been detected in rhizophores of Vernonia herbacea during the phenological cycle. These variations indicate the occurrence of active inulin synthesis and depolymerization throughout the cycle and a role for this carbohydrate as a reserve compound. 1-Fructan exohydrolase (1-FEH) is the enzyme responsible for inulin depolymerization, and its activity has been detected in rhizophores of sprouting plants. Defoliation and low temperature are enhancer conditions of this 1-FEH activity. The aim of the present work was the cloning of this enzyme. Rhizophores were collected from plants induced to sprout, followed by storage at 5 degrees C. A full length 1-FEH cDNA sequence was obtained by PCR and inverse PCR techniques, and expressed in Pichia pastoris. Cold storage enhances FEH gene expression. Vh1-FEH was shown to be a functional 1-FEH, hydrolyzing predominantly beta-2,1 linkages, sharing high identity with chicory FEH sequences, and its activity was inhibited by 81% in the presence of 10 mM sucrose. In V. herbacea, low temperature and sucrose play a role in the control of fructan degradation. This is the first study concerning the cloning and functional analysis of a 1-FEH cDNA of a native species from the Brazilian Cerrado. Results will contribute to understanding the role of fructans in the establishment of a very successful fructan flora of the Brazilian Cerrado, subjected to water limitation and low temperature during winter.

Vernonia amygdalina: anticancer activity, authentication, and adulteration detection.

Evidence suggests that most chemotherapeutic agents are less effective as treatment in patients with estrogen receptor-negative (ER-) breast carcinomas compared to those with estrogen receptor-positive (ER+) breast carcinomas. Moreover, African American Women (AAW) is disproportionately diagnosed with ER- breast cancer compared to their white counterparts. Novel therapies effective against ER- breast carcinomas are urgently needed to ameliorate the health disparity. Previous reports show that low concentrations (microgram/ml) of water-soluble leaf extracts of a Nigerian edible plant, V. amygdalina (VA), potently retards the proliferative activities of ER+ human breast cancerous cells (MCF-7) in vitro in a concentration-dependent fashion. However, the anti-proliferative activities of VA in either ductal or ER- carcinoma cells have not been characterized. The exposure of BT-549 to increasing concentrations of VA (10, 100, and 1000 microg/mL) inhibited cell growth by approximately 14 % (P<0.05), 22 % (p<0.05), and 50 % (p<0.005) respectively. The cell count studies were corroborated by DNA synthesis studies. Treatments of BT-549 with 10, 100, and 1000 microg/mL VA inhibited DNA synthesis in a concentration dependent fashion by 22 %, 76 % (P<0.05), and 86 % (p<0.01) respectively. BT-549 cells were insensitive to 10 and 100 nM paclitaxel (TAX) treatments. Isolation of DNA from dried VA leaves yielded approximately 12.2 and 1 kbp genomic DNA that were Eco RI-insensitive but Hind III and Bam HI-sensitive. These pieces of information may be used to enhance the safety of medicinal botanical VA through authentication, and adulteration detection.