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

An exposition on trichome development and cell shape with a focus on the function of MIXTA-like R2R3-MYBs.

University of Minnesota Ph.D. dissertation. June 2009. Major: Plant Biological Sciences. Advisor: M. David Marks, Ph.D. 1 computer file (PDF); xx, 282 pages.Plant development requires cell differentiation throughout the plant life cycle because plants rely upon the initiation and growth of new organs to reach reproductive maturity. Developmental programs specifying cell pigmentation, cell shape, and specification of cell type have been explored in Arabidopsis. Transcription factors are key components of these developmental programs and work in Arabidopsis and other plant systems have been essential in defining the roles that these factors play during development. A prime example of this in Arabidopsis is the trichome patterning program. The function and structural diversity of trichomes are intimately related, a relationship that this is explored in this thesis. What types of regulatory networks are involved in defining the form of a trichome is visited as well, setting the stage for deeper studies into Arabidopsis trichome development. Use of the glabra 3 shapeshifter (gl3-sst) allele as a proxy for early stages of trichome development in transcriptional profiling reveals the developmental activities of early stage trichomes. Candidate genes from these experiments were then used in a reverse genetics screen to find other genes with trichome phenotypes. Through this method, an R2R3-MYB transcription factor was discovered to play a role in determining cell shape. R2R3-MYB domain transcription factors constitute a major class of transcriptional regulators in plants. The Arabidopsis genome encodes an estimated 125 functional R2R3-MYB proteins. Additionally, R3-MYBs, R1R2R3-MYBs, R-R MYBs, and a single four-repeat MYB protein are encoded by the Arabidopsis genome. Animal genomes only contain a handful of MYB genes. Clearly plants have expanded and utilized this lineage in their evolutionary history, and not surprisingly, many of the regulatory programs these plant genes function in are prominent or specific to plants. As a group, the R2R3-MYB family has been studied previously and authorities have defined various subgroups to which members of this gene class are assigned. This thesis focuses upon members of subgroup 9, defined by the presence of the AQWESA amino acid motif. Seminal work describing the function of this group began with the Antirrhinum majus gene MIXTA. This gene is required for the proper differentiation of conical cells in the floral epidermis. AmMYB MIXTA-like 1, AmMYB MIXTA-like 2, AmMYB MIXTA-like 3, have since been described in Antirrhinum and all have been shown to be functionally similar to MIXTA by heterologus expression in tobacco in the control of cell shape, albeit to varying degrees. Collectively, the available Antirrhinum gene data supports the notion that subgroup 9 R2R3-MYBs are determinants of cell shape be it floral trichomes or conical cells. Here the technical capabilities we possess in Arabidopsis are used to define the function of the subgroup 9 R2R3-MYB NOECK, (NOK, AT3G01140). NOK functions as a negative regulator of trichome branching, leading to trichome cells with increased volume in the mutant line. This phenotype is opposite that of the reduction in cell volume that might occur in mixta Antirrhinum floral epidermal cells that do not become conical by growing out of the epidermal plane. Expression profiling of trichome cells of various mutants including nok revealed coordinately regulated genes that are extracellular matrix components. These findings coupled with the published data indicates that NOK, and perhaps all other subgroup 9 R2R3-MYBs, control cell shape by altering properties of the extracellular matrix. Preliminary data testing the functional equivalence of selected MIXTA-like genes from Antirrhinum majus, Arabidopsis, Dendrobium crumenatum, and Medicago truncatula are given. These data support the portability of the NOK functional characterization data to other plant species. Furthermore, these findings illustrate that subgroup 9 R2R3-MYBs alter cell shape regardless of phylogenic origin.Gilding, Edward Kalani. (2009). An exposition on trichome development and cell shape with a focus on the function of MIXTA-like R2R3-MYBs.. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/54429

Assembly and annotation of a non-model gastropod (Nerita melanotragus) transcriptome: a comparison of De novo assemblers

Background The sequencing, de novo assembly and annotation of transcriptome datasets generated with next generation sequencing (NGS) has enabled biologists to answer genomic questions in non-model species with unprecedented ease. Reliable and accurate de novo assembly and annotation of transcriptomes, however, is a critically important step for transcriptome assemblies generated from short read sequences. Typical benchmarks for assembly and annotation reliability have been performed with model species. To address the reliability and accuracy of de novo transcriptome assembly in non-model species, we generated an RNAseq dataset for an intertidal gastropod mollusc species, Nerita melanotragus, and compared the assembly produced by four different de novo transcriptome assemblers; Velvet, Oases, Geneious and Trinity, for a number of quality metrics and redundancy. Results Transcriptome sequencing on the Ion Torrent PGM™ produced 1,883,624 raw reads with a mean length of 133 base pairs (bp). Both the Trinity and Oases de novo assemblers produced the best assemblies based on all quality metrics including fewer contigs, increased N50 and average contig length and contigs of greater length. Overall the BLAST and annotation success of our assemblies was not high with only 15-19% of contigs assigned a putative function. Conclusions We believe that any improvement in annotation success of gastropod species will require more gastropod genome sequences, but in particular an increase in mollusc protein sequences in public databases. Overall, this paper demonstrates that reliable and accurate de novo transcriptome assemblies can be generated from short read sequencers with the right assembly algorithms. Keywords: Nerita melanotragus; De novo assembly; Transcriptome; Heat shock protein; Ion torren

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

Genetic Transformation and Breeding

Sorghum is a major human staple in the semi-arid tropics, and is also a good model for related species with larger genomes, such as maize and sugarcane. Hence sorghum genome resources have been well-developed. Unfortunately, sorghum genetic transformation has been largely neglected, and has historically lagged behind the other “Big 5" cereals. Until recently, sorghum has been regarded as a largely recalcitrant cereal for biotechnological approaches to its improvement. Thanks to efforts made by transformation teams in Nebraska, California and Queensland, more highly efficient sorghum transformation is now a reality, with good transformation systems available using microprojectiles and Agrobacterium. The future for GM sorghums is now much brighter, and applications for basic biology and the improvement of sorghum agronomically and for specific end-uses is now becoming an integral part of many sorghum improvement programs

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