Butterfly Pea (Clitoria ternatea), a Cyclotide-Bearing Plant With Applications in Agriculture and Medicine

The perennial leguminous herb Clitoria ternatea (butterfly pea) has attracted significant interest based on its agricultural and medical applications, which range from use as a fodder and nitrogen fixing crop, to applications in food coloring and cosmetics, traditional medicine and as a source of an eco-friendly insecticide. In this article we provide a broad multidisciplinary review that includes descriptions of the physical appearance, distribution, taxonomy, habitat, growth and propagation, phytochemical composition and applications of this plant. Notable amongst its repertoire of chemical components are anthocyanins which give C. ternatea flowers their characteristic blue color, and cyclotides, ultra-stable macrocyclic peptides that are present in all tissues of this plant. The latter are potent insecticidal molecules and are implicated as the bioactive agents in a plant extract used commercially as an insecticide. We include a description of the genetic origin of these peptides, which interestingly involve the co-option of an ancestral albumin gene to produce the cyclotide precursor protein. The biosynthesis step in which the cyclic peptide backbone is formed involves an asparaginyl endopeptidase, of which in C. ternatea is known as butelase-1. This enzyme is highly efficient in peptide ligation and has been the focus of many recent studies on peptide ligation and cyclization for biotechnological applications. The article concludes with some suggestions for future studies on this plant, including the need to explore possible synergies between the various peptidic and non-peptidic phytochemicals

Cyclotides in a biotechnological context: opportunities and challenges

Plant biotechnology offers us the ability to augment and improve upon biological systems to produce value added crops, medicines, or specialized products. The application of cyclotides, either naturally occurring or engineered, requires their production at industrial-scale yields. Here we discuss the most relevant applications of cyclotides within a plant biotechnological context and present biological, regulatory, and market factors that should be considered when embarking on a cyclotide production project in planta. It is evident that only a select few plants appear to have evolved to produce cyclotides, as introduced in Chapters 1-4Chapter 1Chapter 2Chapter 3Chapter 4, where they putatively function as highly abundant defense molecules. Their high abundance and stability suggest that they are of primary importance to the species that contain them. Plant biotechnology could be used to harness this functionality and to engineer novel designer cyclotides for expression in plants. We finish with a brief discussion of what the future may hold for cyclotide-based biotechnology

A robust tissue culture system for sorghum [Sorghum bicolor (L.) Moench]

Sorghum tissue culture has been challenged by three predominant obstacles for decades, namely toxic pigments (phenolics), low regeneration frequencies and short duration of callus regenerability. Here, we report a robust tissue culture system for sorghum, which has minimized these major impediments. To optimize media, different concentrations of various plant growth regulators, such as 2,4-dichlorophenoxyacetic acid (2,4-D), N-6-benzyladenine (BA), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and a-naphthaleneacetic acid (NAA) were evaluated. Additional ingredients, including KH2PO4, CuSO4 center dot 5H(2)O, L-asparagine, L-proline and polyvinylpyrrolidone (PVP) were also assessed. Results showed that callus age had a conspicuous effect on its growth and regenerability, with callus weekly growth ratio and regenerability peaked at two weeks after induction. A callus induction rate up to 100% was achieved in inbred line Tx430, whereas regeneration rates up to 100% were obtained from SA281 and 91419R. This highly efficient system has been utilized for sorghum transformation for several years and has been proven to be reliable and reproducible. (C) 2015 SAAB. Published by Elsevier B.V. All rights reserved

Molecular markers in plant improvement

Molecular Markers in Plants surveys an array of technologies used in the molecular analysis of plants. The role molecular markers play in plant improvement has grown significantly as DNA sequencing and high-throughput technologies have matured. This timely review of technologies and techniques will provide readers with a useful resource on the latest molecular technologies. Molecular Markers in Plants not only reviews past achievements, but also catalogs recent advances and looks forward towards the future application of molecular technologies in plant improvement. Opening chapters look at the development of molecular technologies. Subsequent chapters look at a wide range of applications for the use of these advances in fields as diverse as plant breeding, production, biosecurity, and conservation. The final chapters look forward toward future developments in the field. Looking broadly at the field of molecular technologies, Molecular Markers in Plants will be an essential addition to the library of every researcher, institution, and company working in the field of plant improvement. [Book Synopsis

The role of pullulanase in starch biosynthesis, structure, and thermal properties by studying sorghum with increased pullulanase activity

Pullulanase is a starch debranching enzyme involved in starch biosynthesis, but its function in starch biosynthesis is not fully established. This study aims to understand its function by analyzing a sorghum variety (SbPUL-RA) with increased pullulanase activity (67% higher than wild-type). The results demonstrate that increased pullulanase activity has little or no effect on crystalline structure or amylose content; however, it increases the total amount of starch, and produces amylose with longer chains. The changed amylose structures results in slightly decreased starch gelatinization temperatures. These results indicate a hitherto unconsidered role for pullulanase during amylose biosynthesis: it removes shorter amylose branches, which further facilitate the elongation of existing amylose chains

Increasing protein content and digestibility in sorghum grain with a synthetic biology approach

Despite great genetic diversity, sorghum grain consistently suffers from poor protein digestibility. The physicochemical packaging of protein bodies which consist of protease-resistant β- and γ-kafirin is considered a major obstacle. A synthetic β-kafirin gene, which shares the endosperm-specific promoter and signal peptide with the native β-kafirin gene (Sobic.009G001600.1), was transformed into sorghum inbred line Tx430. The gene was modified with ten additional proteolytic sites. These sites were designed to be amenable to cleavage by pepsin and/or chymotrypsin proteinases. Five independent transgenic lines were regenerated by microprojectile transformation. Notably, considerably more protein was observed in the peripheral endosperm of transgenic lines under scanning electron microscopy. Microscopy revealed invaginated or irregularly shaped protein bodies in the endosperm of transgenic lines. Grains of transgenic lines contained 11–37% more protein, which was 11–21% more pepsin digestible and 7–25% more chymotrypsin digestible than Tx430. Additionally, the abundant synthetic β-kafirin protein (5.6% of total protein) was detected by mass spectrometry data analysis in the transgenic line 9-1. Field-grown homozygous transgenics retained higher protein content, larger seed size and no reduction in grain number per plant. The results illustrated that plant synthetic biology could play an important role in improving sorghum nutritional value

Insecticidal diversity of butterfly pea (Clitoria ternatea) accessions

Butterfly pea (Clitoria ternatea) is currently the only leguminous plant species known to produce a suite of ultrastable cyclic plant defense peptides called cyclotides. For agricultural applications, cyclotides have attracted significant interest, leading to the recent registration of a butterfly pea extract as an ecofriendly pesticide (Sero-X®). In this study, we set out to distinguish the variation in cyclotide expression and toxicity towards insect cells for butterfly pea accessions sourced worldwide. In characterizing the peptide extracts from 23 butterfly pea accessions sourced from 11 countries, we show that significant variation in cyclotide expression exists between them. For some accessions, the cyclotide Cter M, typically the most abundantly expressed cyclotide in vegetative butterfly pea tissues, is absent. Genomic and transcriptomic sequencing revealed the presence of CterM-like precursor genes in these accessions that contained missense mutations that were likely contributing to the lack of Cter M expression. Peptide profiling also showed that one accession does not produce detectable levels of other cyclotides: cliotide T1, cliotide T4, Cter A and Cter Q. A comparison of cytotoxicity against Sf9 (Spodoptera frugiperda) cells revealed that cytotoxicity is not dependent on Cter M, with accessions lacking this peptide also displaying cytotoxicity. Overall, insights from this study provides foundational knowledge about characters to be considered for selective breeding of butterfly pea with enhanced insecticidal properties

The functionality of α-kafirin promoter and α-kafirin signal peptide

Cereal grains offer great potential as a storage system for production of highly valuable proteins using biotechnological approaches, but such applications require tight temporal and spatial control of transgene expression. Towards this aim, we have undertaken a detailed analysis of α-kafirin (α-kaf) promoter and α-kaf signal peptide (sp) in transgenic sorghum plants, using green fluorescent protein gene (gfp) as a reporter. Constructs containing either the α-kaf promoter or the constitutive maize ubiquitin-1 (ubi) promoter driving either gfp or sp-gfp translational fusion were introduced into Sorghum bicolor inbred line Tx430 by particle bombardment. We show for the first time that the α-kaf promoter directs endosperm-specific transgene expression, with activity first detected at 10\ua0days post-anthesis (dpa), peaking at 20 dpa, and remaining active through to physiological maturity. Furthermore, we demonstrate for the first time that the α-kafirin sp is sufficient to direct foreign protein to protein bodies in the endosperm. The evidence is also provided for possible mis-targeting by α-kaf sp in vegetative tissues of transgenic lines with ubi-sp-gfp, resulting in loss of reporter gene translational activity that no GFP signal was observed. These results demonstrate that α-kaf promoter and α-kaf sp are well suited for seed bioengineering to produce recombinant proteins in sorghum endosperm or deposit foreign proteins into sorghum protein bodies

Bayesian nonparametric sparse seemingly unrelated regression model (SUR)

Seemingly unrelated regression (SUR) models are used in studying the interactions among economic variables of interest. In a high dimensional setting and when applied to large panel of time series, these models have a large number of parameters to be estimated and suffer of inferential problems. We propose a Bayesian nonparametric hierarchical model for multivariate time series in order to avoid the overparametrization and overfitting issues and to allow for shrinkage toward multiple prior means with unknown location, scale and shape parameters. We propose a two-stage hierarchical prior distribution. The first stage of the hierarchy consists in a lasso conditionally independent prior distribution of the Normal-Gamma family for the SUR coefficients. The second stage is given by a random mixture distribution for the Normal-Gamma hyperparameters, which allows for parameter parsimony through two components. The first one is a random Dirac point-mass distribution, which induces sparsity in the SUR coefficients; the second is a Dirichlet process prior, which allows for clustering of the SUR coefficients. We provide a Gibbs sampler for posterior approximations based on introduction of auxiliary variables. Some simulated examples show the efficiency of the proposed. We study the effectiveness of our model and inference approach with an application to macroeconomics

Circular permutation of the native enzyme-mediated cyclization position in cyclotides

Cyclotides are a class of cyclic disulfide-rich peptides found in plants that have been adopted as a molecular scaffold for pharmaceutical applications due to their inherent stability and ability to penetrate cell membranes. For research purposes, they are usually produced and cyclized synthetically, but there are concerns around the cost and environmental impact of large-scale chemical synthesis. One strategy to improve this is to combine a recombinant production system with native enzyme-mediated cyclization. Asparaginyl endopeptidases (AEPs) are enzymes that can act as peptide ligases in certain plants to facilitate cyclotide maturation. One of these ligases, OaAEP1b, originates from the cyclotide-producing plant, , and can be produced recombinantly for use as an alternative to chemical cyclization of recombinant substrates. However, not all engineered cyclotides are compatible with AEP-mediated cyclization because new pharmaceutical epitopes often replace the most flexible region of the peptide, where the native cyclization site is located. Here we redesign a popular cyclotide grafting scaffold, MCoTI-II, to incorporate an AEP cyclization site located away from the usual grafting region. We demonstrate the incorporation of a bioactive peptide sequence in the most flexible region of MCoTI-II while maintaining AEP compatibility, where the two were previously mutually exclusive. We anticipate that our AEP-compatible scaffold, based on the most popular cyclotide for pharmaceutical applications, will be useful in designing bioactive cyclotides that are compatible with AEP-mediated cyclization and will therefore open up the possibility of larger scale enzyme-mediated production of recombinant or synthetic cyclotides alike