A vicilin-like protein extracted from Clitoria fairchildiana cotyledons was toxic to Callosobruchus maculatus (Coleoptera: Chrysomelidae).

Callosobruchus maculatus is the main pest cowpea (Vigna unguiculata). Given its relevance as an insect pest, studies have focused in finding toxic compounds which could prevent its predatory action towards the seeds. Clitoria fairchildiana is a native Amazon species, whose seeds are refractory to insect predation. This characteristic was the basis of our interest in evaluating the toxicity of its seed proteins to C. maculatus larvae. Seed proteins were fractioned, according to their solubility, to albumins (F1), globulins (F2), kaphyrins (F3), glutelins (F4), linked kaphyrins (F5) and cross-linked glutelins (F6). The fractionated proteins were quantified, analysed by tricine-SDS-PAGE and inserted into the diet of this insect pest in order to evaluate their insecticidal potential. The most toxic fraction to C. maculatus, the propanol soluble F3, was submitted to molecular exclusion chromatography and all of the peaks obtained, F3P1, F3P2, F3P3, caused a reduction of larval mass, especially F3P1, seen as a major ~12 kDa electrophoretic band. This protein was identified as a vicilin-like protein by mass spectrometry and BLAST analysis. The alignment of the Cfvic (C. fairchildiana vicilin) peptides with a V. unguiculata vicilin sequence, revealed that Cfvic has at least five peptides (ALLTLVNPDGR, AILTLVNPDGR, NFLAGGKDNV, ISDINSAMDR, NFLAGEK) which lined up with two chitin binding sites (ChBS). This finding was corroborated by chitin affinity chromatography and molecular docking of chitin-binding domains for N-Acetyl-D-glucosamine and by the reduction of Cfvic chitin affinity after chemical modification of its Lys residues. In conclusion, Cfvic is a 12 kDa vicilin-like protein, highly toxic to C. maculatus, acting as an insect toxin through its ability to bind to chitin structures present in the insect midgut.

Study on activation mechanism and cleavage sites of recombinant butelase-1 zymogen derived from Clitoria ternatea.

Asparagine endopeptidases (AEPs) were synthesized as a zymogen and were known to undergo pH-dependent autoproteolytic activation using their endopeptidase activity. Butelase-1, one of the few AEPs with ligation activity, can also be synthesized as a zymogen and activated at acidic pH in vitro, but the detailed activation process and potential activation sites of its zymogen are not fully understood. In this study, recombinant butelase-1 exhibited high ligation activity and ineffective endopeptidase activity, and its activities were strictly pH-dependent. The endopeptidase activity caused the activation of butelase-1 zymogen at acidic pH, which was autocatalytic, required sequential removal of C- and N-terminal pro-peptides, and was a bimolecular reaction. The pro-peptides were critical to the stability of butelase-1. Once the pro-peptides left the active domain, butelase-1 was quickly inactivated at pH 7.0. Based on the LC-MS/MS sequencing of activation products, Asp319 and Asn322 were identified as potential C-terminal pro-region hydrolysis sites of the butelase-1 zymogen, which was validated by site-directed mutagenesis. Our results provided a reasonable explanation for the self-activation of butelase-1 zymogen in vitro and provided supplementary information for the activation of AEP ligase zymogen.

Disulfide-Rich Cyclic Peptides from Clitoria ternatea Protect against ß-Amyloid Toxicity and Oxidative Stress in Transgenic Caenorhabditis elegans.

Neurotoxic aggregation of ß-amyloid (Aß) peptides is a hallmark of Alzheimer's disease and increased reactive oxygen species (ROS) is an associated process. In the present study, we report the neuroprotective effects of disulfide-rich, circular peptides from Clitoria ternatea (C. ternatea) (butterfly pea) on Aß-induced toxicity in transgenic Caenorhabditis elegans. Cyclotides (∼30 amino acids long) are a special class of cyclic cysteine knot peptides. We show that cyclotide-rich fractions from different plant tissues delay Aß-induced paralysis in the transgenic CL4176 strain expressing the human muscle-specific Aß1-42 gene. They also improved Aß-induced chemotaxis defects in CL2355 strain expressing Aß1-42 in the neuronal cells. ROS assay suggests that this protection is likely mediated by the inhibition of Aß oligomerization. Furthermore, Aß deposits were reduced in the CL2006 strain treated with the fractions. The study shows that cyclotides from C. ternatea could be a source of a novel pharmacophore scaffold against neurodegenerative diseases.

Transcriptomic profiling of the medicinal plant Clitoria ternatea: identification of potential genes in cyclotide biosynthesis.

Clitoria ternatea a perennial climber of the Fabaceae family, is well known for its agricultural and medical applications. It is also currently the only known member of the Fabaceae family that produces abundant amounts of the ultra-stable macrocyclic peptides, cyclotides, across all tissues. Cyclotides are a class of gene-encoded, disulphide-rich, macrocyclic peptides (26-37 residues) acting as defensive metabolites in several plant species. Previous transcriptomic studies have demonstrated the genetic origin of cyclotides from the Fabaceae plant family to be embedded in the albumin-1 genes, unlike its counterparts in other plant families. However, the complete mechanism of its biosynthesis and the repertoire of enzymes involved in cyclotide folding and processing remains to be understood. In this study, using RNA-Seq data and de novo transcriptome assembly of Clitoria ternatea, we have identified 71 precursor genes of cyclotides. Out of 71 unique cyclotide precursor genes obtained, 51 sequences display unique cyclotide domains, of which 26 are novel cyclotide sequences, arising from four individual tissues. MALDI-TOF mass spectrometry analysis of fractions from different tissue extracts, coupled with precursor protein sequences obtained from transcriptomic data, established the cyclotide diversity in this plant species. Special focus in this study has also been on identifying possible enzymes responsible for proper folding and processing of cyclotides in the cell. Transcriptomic mining for oxidative folding enzymes such as protein-disulphide isomerases (PDI), ER oxidoreductin-1 (ERO1) and peptidylprolyl cis-trans isomerases (PPIases)/cyclophilins, and their levels of expression are also reported. In particular, it was observed that the CtPDI genes formed plant-specific clusters among PDI genes as compared to those from other plant species. Collectively, this work provides insights into the biogenesis of the medicinally important cyclotides and establishes the expression of certain key enzymes participating in peptide biosynthesis. Also, several novel cyclotide sequences are reported and precursor sequences are analysed in detail. In the absence of a published reference genome, a comprehensive transcriptomics approach was adopted to provide an overview of diverse properties and constituents of C. ternatea.

Clitoria ternatea L. petal bioactive compounds display antioxidant, antihemolytic and antihypertensive effects, inhibit α-amylase and α-glucosidase activities and reduce human LDL cholesterol and DNA induced oxidation.

The purpose of this study was to use a statistical approach to optimise the experimental conditions regarding the extraction of bioactive compounds, and to analyse the in vitro functional properties of crude lyophilized extracts (CLE) and partially purified (PPE) extracts of Clitoria ternatea petals. The results showed that the factors of temperature and time influenced the extraction of phenolic compounds, antioxidant activity and the physicochemical parameters. Simultaneous optimisation showed that the same levels of bioactive compounds were extracted when using temperatures from 11.7 to 68.3 °C and times from 8.47 to 51.12 min. Principal component analysis revealed the experimental conditions that provided the extraction producing the highest level of phenolic content (40 °C/30 min). The CLE showed antimicrobial activity; protective effect against hemolysis of erythrocytes; inhibition of α-amylase, α-glucosidase and angiotensin-I-converting (ACE-I) enzymes; and inhibition of lipid peroxidation. The CLE and PPE demonstrated oxygen radical absorption capacity; inhibition of DNA strand scission; inhibition of LDL cholesterol oxidation; intracellular antioxidant activity against reactive oxygen species (>100 µg/mL); and no cytotoxicity (IC50, GI50 and LC50 > 900 µg/mL) against A549, HCT8 and IMR90 cell lines.

Inoculated Clitoria ternatea with Bacillus cereus ERBP for enhancing gaseous ethylbenzene phytoremediation: Plant metabolites and expression of ethylbenzene degradation genes.

Air pollutants especially polyaromatic hydrocarbons pose countless threats to the environment. This issue demands for an effective phytoremediation technology. In this study we report the beneficial interactions of Clitoria ternatea and its plant growth promoting endophytic bacteria Bacillus cereus ERBP by inoculating it for the remediation of 5 ppm airborne ethylbenzene (EB). The percentage efficiency for ethylbenzene removal among B. cereus ERBP inoculated and non-inoculated sterile and natural C. ternatea has also been determined. The inoculation of B. cereus ERBP has significantly increased EB removal efficiency of both sterile and natural C. ternatea. The inoculated natural C. ternatea seedlings showed 100% removal efficiency within 84 h for the aforementioned pollutant compared with the sterile inoculated C. ternatea seedlings (108 h). The degradation of EB by C. ternatea seedlings with and without B. cereus ERBP was assessed by measuring the intermediates of EB including 1-phenylethanol, acetophenon, benzaldehyde and benzoic acid. In addition, cytochrome P450s monooxygenase (CYP83D1) and dehydrogenases (LOC100783159) involved in the oxidation of hydrocarbons are well reported for their bio catalytic activities under xenobiotic stress conditions. Hence, the co-effect of the native endophyte B. cereus ERBP inoculation and EB exposure on the expression level of CYP83D1 and dehydrogenase were also determined. The targeted genes CYP83D1and dehydrogenases have shown an increased expression level under the 5 ppm of EB exposure enabling C. ternatea to withstand and remediate the pollutant.

Peptidomic Identification of Cysteine-Rich Peptides from Plants.

Plant cysteine-rich peptides (CRPs) constitute a majority of plant-derived peptides with high molecular diversity. This protocol describes a rapid and efficient peptidomic approach to identify a whole spectrum of CRPs in a plant extract and decipher their molecular diversity and bioprocessing mechanism. Cyclotides from C. ternatea are used as the model CRPs to demonstrate our methodology. Cyclotides exist naturally in both cyclic and linear forms, although the linear forms (acyclotide) are generally present at much lower concentrations. Both cyclotides and acyclotides require linearization of their backbone prior to fragmentation and sequencing. A novel and practical three-step chemoenzymatic treatment was developed to linearize and distinguish both forms: (1) N-terminal acetylation that pre-labels the acyclotides; (2) conversion of Cys into pseudo-Lys through aziridine-mediated S-alkylation to reduce disulfide bonds and to increase the net charge of peptides; and (3) opening of cyclic backbones by the novel asparaginyl endopeptidase butelase 2 that cleaves at the native bioprocessing site. The treated peptides are subsequently analyzed by liquid chromatography coupled to mass spectrometry using electron transfer dissociation fragmentation and sequences are identified by matching the MS/MS spectra directly with the transcriptomic database.

Influence of soil cover and N and K fertilization on the quality of biofortified QPM in the humid tropics.

BACKGROUND: In the humid tropics, unfavorable conditions present challenges to smallholder farmers attempting to meet food demands. The objective of this study was to evaluate the influence of alley cropping and addition of potassium and nitrogen on the productivity and nutritional value of quality protein maize (QPM). The experimental design consisted of randomized blocks with four replicates in a 5 × 2 factorial scheme, with five treatments, Gliricidia + Acacia (GA), Gliricidia + Clitoria (GC), Leucaena + Acacia (LA), Leucaena + Clitoria (LC) and bare soil (BS), in two cropping systems, one with addition of nitrogen and potassium (NK) and one without. RESULTS: The grain yield of LC + NK was significantly higher than that of all other treatments except GC + NK and LA + NK, and six times higher than that of BS + NK. The protein content of LC + NK was higher than that of the treatments without residue. CONCLUSION: Although the mulching of tree legumes increased the yield and quality of food for smallholder agriculture, achieving this outcome requires eliminating potentially negative interactions when combining trees and crops in addition to enhancing the availability and uptake of nutrients. © 2015 Society of Chemical Industry.

Hairy root cultures of butterfly pea (Clitoria ternatea L.): Agrobacterium × plant factors influencing transformation.

Transformed rhizoclones were developed from Agrobacterium-treated explants of the medicinally important twinning legume Clitoria ternatea L. Several key factors influencing transformation events were optimized. A4T was the most infectious among the strains employed. Internode segments were more responsive than leaves, outdoor-grown explants preferred to those from in vitro cultures. High frequency transformation, resulting in up to 85.8% rhizogenesis, was attained using pre-pricked internodal explants for immersion (10 min) in Agrobacterium rhizogenes suspension grown overnight with acetosyringone (100 µM) to an OD(660) â‰… 0.6, diluted to a density of 10(9) cells ml(-1), followed by 5-day co-cultivation. Roots were individually cultured in MS0 supplemented with the bacteriostatic antibiotic cefotaxime (500 µg ml(-1)). Rhizoclones were renewed through successive subcultures in MS0 under diffused illumination. The T ( L )-DNA rolB and rolC ORF were detected in rhizoclones through PCR amplification. The T ( R )-DNA gene encoding mannopine synthase (man2) was revealed by positive amplification and opine gene expression substantiated by agropine and mannopine biosynthesis in all selected transformed rhizoclones. The implication of such findings is discussed on the context of utilization of such genetically transformed root cultures towards sustainable production of medicinally useful phytocompounds, besides providing a means for plant conservation.

Discovery and characterization of novel cyclotides originated from chimeric precursors consisting of albumin-1 chain a and cyclotide domains in the Fabaceae family.

The tropical plant Clitoria ternatea is a member of the Fabaceae family well known for its medicinal values. Heat extraction of C. ternatea revealed that the bioactive fractions contained heat-stable cysteine-rich peptides (CRPs). The CRP family of A1b (Albumin-1 chain b/leginsulins), which is a linear cystine knot CRP, has been shown to present abundantly in the Fabaceae. In contrast, the cyclotide family, which also belongs to the cystine knot CRPs but with a cyclic structure, is commonly found in the Rubiaceae, Violaceae, and Cucurbitaceae families. In this study, we report the discovery of a panel of 15 heat-stable CRPs, of which 12 sequences (cliotide T1-T12) are novel. We show unambiguously that the cliotides are cyclotides and not A1bs, as determined by their sequence homology, disulfide connectivity, and membrane active properties indicated by their antimicrobial activities against Escherichia coli and cytotoxicities to HeLa cells. We also show that cliotides are prevalent in C. ternatea and are found in every plant tissue examined, including flowers, seeds, and nodules. In addition, we demonstrate that their precursors are chimeras, half from cyclotide and the other half from Albumin-1, with the cyclotide domain displacing the A1b domain in the precursor. Their chimeric structures likely originate from either horizontal gene transfer or convergent evolution in plant nuclear genomes, which are exceedingly rare events. Such atypical genetic arrangement also implies a different mechanism of biosynthetic processing of cyclotides in the Fabaceae and provides new understanding of their evolution in plants.

Biosynthesis of malonylated flavonoid glycosides on the basis of malonyltransferase activity in the petals of Clitoria ternatea.

The crude malonyltransferase from the petals of Clitoria ternatea was characterized enzymatically to investigate its role on the biosynthetic pathways of anthocyanins and flavonol glycosides. In C. ternatea, a blue flower cultivars (DB) and mauve flower variety (WM) accumulate polyacylated anthocyanins (ternatins) and delphinidin 3-O-(6''-O-malonyl)-beta-glucoside which is one of the precursors of ternatins, respectively. Moreover, WM accumulates minor delphinidin glycosides - 3-O-beta-glucoside, 3-O-(2''-O-alpha-rhamnosyl)-beta-glucoside, 3-O-(2''-O-alpha-rhamnosyl-6''-O-malonyl)-beta-glucoside of delphinidin. These glycosidic patterns for minor anthocyanins in WM are also found among the minor flavonol glycosides in all the varieties including a white flower variety (WW) although the major flavonol glycosides are 3-O-(2''-O-alpha-rhamnosyl)-beta-glucoside, 3-O-(6''-O-alpha-rhamnosyl)-beta-glucoside, 3-O-(2'',6''-di-O-alpha-rhamnosyl)-beta-glucoside of kaempferol, quercetin, and myricetin. How do the enzymatic characteristics affect the variety of glycosidic patterns in the flavonoid glycoside biosynthesis among these varieties? While the enzyme from DB highly preferred delphinidin 3-O-beta-glucoside in the presence of malonyl-CoA, it also has a preference for other anthocyanidin 3-O-beta-glucosides. It could use flavonol 3-O-beta-glucosides in much lower specific activities than anthocyanins; however, it could not utilize 3-O-(2''-O-alpha-rhamnosyl)-beta-glucosides of anthocyanins and flavonols, and 3,3'-di- and 3,3',5'-tri-O-beta-glucoside of delphinidin - other possible precursors in ternatins biosynthesis. It highly preferred malonyl-CoA as an acyl donor in the presence of delphinidin 3-O-beta-glucoside. The crude enzymes prepared from WM and WW had the same enzymatic characteristics. These results suggested that 3-O-(2''-O-alpha-rhamnosyl-6''-O-malonyl)-beta-glucosides of flavonoids were synthesized via 3-O-(6''-O-malonyl)-beta-glucosides rather than via 3-O-(2''-O-alpha-rhamnosyl)-beta-glucosides, and that malonylation proceeded prior to glucosylation at the B-ring of delphinidin in the early biosynthetic steps towards ternatins. It seemed that the substrate specificities largely affected the difference in the accumulated amount of malonylated glycosides between anthocyanins and flavonols although they are not simply proportional to the accumulation ratio. This enzyme might join in the production of both malonylanthocyanins and flavonol malonylglycosides as a result of broad substrate specificities towards flavonoid 3-O-beta-glucosides.