Organellar carbon metabolism is co-ordinated with distinct developmental phases of secondary xylem

Subcellular compartmentation of plant biosynthetic pathways in the mitochondria and plastids requires coordinated regulation of nuclear encoded genes, and the role of these genes has been largely ignored by wood researchers. In this study, we constructed a targeted systems genetics coexpression network of xylogenesis in Eucalyptus using plastid and mitochondrial carbon metabolic genes and compared the resulting clusters to the aspen xylem developmental series. The constructed network clusters reveal the organization of transcriptional modules regulating subcellular metabolic functions in plastids and mitochondria. Overlapping genes between the plastid and mitochondrial networks implicate the common transcriptional regulation of carbon metabolism during xylem secondary growth. We show that the central processes of organellar carbon metabolism are distinctly coordinated across the developmental stages of wood formation and are specifically associated with primary growth and secondary cell wall deposition. We also demonstrate that, during xylogenesis, plastid-targeted carbon metabolism is partially regulated by the central clock for carbon allocation towards primary and secondary xylem growth, and we discuss these networks in the context of previously established associations with wood-related complex traits. This study provides a new resolution into the integration and transcriptional regulation of plastid- and mitochondrial-localized carbon metabolism during xylogenesis

Functional genomics and systems genetics of cellulose biosynthesis in Eucalyptus

The globally emerging bioeconomy demands rapid advancement in the sustainable production and utilization of bio-based raw materials for a multitude of downstream applications, particularly in the areas of food, health and bioenergy and biomaterials. These needs, particularly pertaining to plant productivity, quality and stress tolerance, will need to be addressed with advanced biotechnology strategies, which accelerate progress beyond what has been achieved with traditional breeding and cultivation methods. Woody biomass is a readily available source of renewable carbon, and trees from the genus Eucalyptus, displaying superior growth and wood properties and established agricultural practices worldwide, are attractive candidates as short-rotation (5-9 years) feedstocks for biofuels and biomaterials. Guiding advanced strategies in biotechnology in Eucalyptus and other biomass feedstocks requires a sophisticated understanding of the molecular underpinnings of carbon allocation and cell wall biology. In the work presented here, we aimed to characterize the molecular biology of cellulose biosynthesis in Eucalyptus xylem (developing wood) and identify genes, processes and pathways that are linked to and possibly influence this process. We achieved this by detailed characterization of field-grown Eucalyptus hybrid trees, utilizing RNA-sequencing technology and metabolomics of xylem as well as measuring wood properties that are thought to impact the efficiency of industrial processing. Given the lack of information with regards to gene expression in Eucalyptus trees, a major aim was to characterize transcriptomes from various tissues and organs, including a cellulose-enriched form of xylem called tension wood. This involved challenging bioinformatics, which resulted in a high quality assembly and publication of a comprehensive gene catalogue for Eucalyptus, which was one of the first short-read RNA-sequencing based de novo assembly from a eukaryotic organism. We also characterized and modelled the properties of cellulose and xylan biosynthetic pathways as a biological system, the parts of which are segregating in Eucalyptus hybrid tree populations, which has generated novel insights into the allocation and partitioning of sequestered carbon between cellulose, xylan and lignin during active secondary cell wall deposition in woody stem tissues. This research has made important contributions to the field of Eucalyptus biology, but also to the broader field of secondary cell wall biosynthesis in plants, specifically providing (i) resources for transcriptome analysis in a large woody perennial (ii) new biological insight into carbon allocation for polysaccharide biosynthesis in wood, and (iii) annotation and discovery of candidate genes and pathways that may influence wood chemical composition and structures. Importantly, we find that cellulose and xylan biosynthetic genes are transcriptionally hardwired in their co-regulation (along with other important processes for cellulose and xylan transport and deposition), likely due to the fact that they utilize a common source of sucrose-derived carbon for cell wall biosynthesis and the production of sufficient energy to do so. This co-regulation appears to be distinct from the regulation of other cell wall biopolymers. Furthermore, evidence from xylem gene expression and metabolite availability in xylem, as research has made important contributions to the field of Eucalyptus biology, but also to the broader field of secondary cell wall biosynthesis in plants, specifically providing (i) resources for transcriptome analysis in a large woody perennial (ii) new biological insight into carbon allocation for polysaccharide biosynthesis in wood, and (iii) annotation and discovery of candidate genes and pathways that may influence wood chemical composition and structures. Importantly, we find that cellulose and xylan biosynthetic genes are transcriptionally hardwired in their co-regulation (along with other important processes for cellulose and xylan transport and deposition), likely due to the fact that they utilize a common source of sucrose-derived carbon for cell wall biosynthesis and the production of sufficient energy to do so. This co-regulation appears to be distinct from the regulation of other cell wall biopolymers. Furthermore, evidence from xylem gene expression and metabolite availability in xylem, as well as from wood properties of field-grown trees, supports a model in which sucrose-derived cytosolic fructose is shunted to the production of lignin precursors during cellulose and xylan biosynthesis. This model parsimoniously explains a mechanism for trees to partition carbon between polysaccharide and lignin synthesis, and provides exciting new questions and potential strategies to influence carbon allocation in the secondary cell walls of woody plants.Thesis (PhD)--University of Pretoria, 2013.Wood and Fibre Molecular Genetics (WFMG) ProgrammeNational Research Foundation (NRF)Technology and Human Resources for Industry Programme (THRIP)GeneticsPhDUnrestricte

Solubility, particle formation and immune display of trimers of major capsid protein 7 of African horsesickness virus fused with enhanced green fluorescent protein

Modified Viral Protein 7 (VP7) of African horsesickness virus (AHSV) is being investigated as a peptide display protein. The protein represents a good candidate for recombinant peptide display due to its tertiary structure, which contains flexible hydrophilic loops on the top domain of the protein where small peptides can potentially be inserted. In addition, wild type (WT) AHSV VP7 tends to form hexagonal crystals of predictable shape and size when expressed in a recombinant expression system. Previous research has resulted in a number of AHSV VP7 genes containing modified cloning sites where DNA representing immunologically relevant peptides can be inserted. When these chimeric proteins are expressed the peptides should be displayed on the surface of the VP7 platform. Several studies have tested the ability to insert peptides of varying lengths into these sites and successfully express the chimeric protein. In these past cases, successful expression of a recombinant chimeric protein was gauged by the observation of particles formed by multimers of VP7 proteins that resemble the one formed by WT-VP7. However, little is known about the ability of these chimeric proteins to act as successful peptide presentations vectors. Specifically, it is not known whether the fusion peptides would retain their correct tertiary structure, or indeed be displayed to the surrounding environment in order to generate a specific immune response. Furthermore, there has been no investigation to track these chimeric proteins’ expression in a heterologous expression system. This dissertation attempts to answer several of these questions through the use of a fluorescent protein, enhanced green fluorescent protein (eGFP), as a model peptide. The use of eGFP as a model peptide can prove correct tertiary structure of the fusion peptide via function of the protein (fluorescence), as well as act as a means of monitoring expression of chimeric VP7-eGFP proteins. Chapter 1 of this dissertation reviews literature that is relevant to AHSV VP7 and the use of fluorescent proteins as fluorescent markers. In addition, the recombinant expression of proteins is discussed, with a focus on solubility and expression levels of expressed proteins. Next, a brief overview is given with regards to vaccination strategies that can be undertaken, with a focus on subunit vaccines and their viability as successful alternatives to live-attenuated vaccines. Finally, the progress with regards to using modified AHSV VP7 as a peptide presentation vector is discussed. Chapter 2 investigates the chimeric protein VP7-177-eGFP, including its construction via a recombinant DNA cloning strategy, its expression in Insect cells using a recombinant Baculovirus expression system, and the ability of eGFP to act as a model fusion peptide on the top domain of a modified VP7 protein. Several experiments investigate whether the chimeric protein maintains its tertiary structure under a series of purification steps, and investigate the solubility of the chimeric protein throughout the expression cycle. Finally, purified forms of the chimeric protein are examined for their ability to generate an immune response specific to the fusion protein, eGFP.<pDissertation (MSc)--University of Pretoria, 2011.Geneticsunrestricte

Evidence for an ancient whole genome duplication in the cycad lineage

Contrary to the many whole genome duplication events recorded for angiosperms (flowering plants), whole genome duplications in gymnosperms (non-flowering seed plants) seem to be much rarer. Although ancient whole genome duplications have been reported for most gymnosperm lineages as well, some are still contested and need to be confirmed. For instance, data for ginkgo, but particularly cycads have remained inconclusive so far, likely due to the quality of the data available and flaws in the analysis. We extracted and sequenced RNA from both the cycad Encephalartos natalensis and Ginkgo biloba. This was followed by transcriptome assembly, after which these data were used to build paralog age distributions. Based on these distributions, we identified remnants of an ancient whole genome duplication in both cycads and ginkgo. The most parsimonious explanation would be that this whole genome duplication event was shared between both species and had occurred prior to their divergence, about 300 million years ago

Loss of wood formation genes in monocot genomes

Woodiness (secondary xylem derived from vascular cambium) has been gained and lost multiple times in the angiosperms, but has been lost ancestrally in all monocots. Here, we investigate the conservation of genes involved in xylogenesis in fully sequenced angiosperm genomes, hypothesising that monocots have lost some essential orthologs involved in this process. We analysed the conservation of genes preferentially expressed in the developing secondary xylem of two eudicot trees in the sequenced genomes of 26 eudicot and seven monocot species, and the early-diverging angiosperm Amborella trichopoda. We also reconstructed a regulatory model of early vascular cambial cell identity and differentiation and investigated the conservation of orthologs across the angiosperms. Additionally, we analysed the genome of the aquatic seagrass Zostera marina for additional losses of genes otherwise essential to, especially, secondary cell wall formation. Despite almost complete conservation of orthology within the early cambial differentiation gene network, we show a clear pattern of loss of genes preferentially expressed in secondary xylem in the monocots that are highly conserved across eudicot species. Our study provides candidate genes that may have led to the loss of vascular cambium in the monocots, and, by comparing terrestrial angiosperms to an aquatic monocot, highlights genes essential to vasculature on land

De novo assembled expressed gene catalog of a fast-growing Eucalyptus tree produced by Illumina mRNA-Seq

<p>Abstract</p> <p>Background</p> <p><it>De novo </it>assembly of transcript sequences produced by short-read DNA sequencing technologies offers a rapid approach to obtain expressed gene catalogs for non-model organisms. A draft genome sequence will be produced in 2010 for a <it>Eucalyptus </it>tree species (<it>E. grandis</it>) representing the most important hardwood fibre crop in the world. Genome annotation of this valuable woody plant and genetic dissection of its superior growth and productivity will be greatly facilitated by the availability of a comprehensive collection of expressed gene sequences from multiple tissues and organs.</p> <p>Results</p> <p>We present an extensive expressed gene catalog for a commercially grown <it>E. grandis </it>× <it>E. urophylla </it>hybrid clone constructed using only Illumina mRNA-Seq technology and <it>de novo </it>assembly. A total of 18,894 transcript-derived contigs, a large proportion of which represent full-length protein coding genes were assembled and annotated. Analysis of assembly quality, length and diversity show that this dataset represent the most comprehensive expressed gene catalog for any <it>Eucalyptus </it>tree. mRNA-Seq analysis furthermore allowed digital expression profiling of all of the assembled transcripts across diverse xylogenic and non-xylogenic tissues, which is invaluable for ascribing putative gene functions.</p> <p>Conclusions</p> <p><it>De novo </it>assembly of Illumina mRNA-Seq reads is an efficient approach for transcriptome sequencing and profiling in <it>Eucalyptus </it>and other non-model organisms. The transcriptome resource (Eucspresso, <url>http://eucspresso.bi.up.ac.za/</url>) generated by this study will be of value for genomic analysis of woody biomass production in <it>Eucalyptus </it>and for comparative genomic analysis of growth and development in woody and herbaceous plants.</p

Horsetails are ancient polyploids : evidence from Equisetum giganteum

Horsetails represent an enigmatic clade within the land plants. Despite consisting only of one genus (Equisetum) that contains 15 species, they are thought to represent the oldest extant genus within the vascular plants dating back possibly as far as the Triassic. Horsetails have retained several ancient features and are also characterized by a particularly high chromosome count (n = 108). Whole-genome duplications (WGDs) have been uncovered in many angiosperm clades and have been associated with the success of angiosperms, both in terms of species richness and biomass dominance, but remain understudied in nonangiosperm clades. Here, we report unambiguous evidence of an ancient WGD in the fern linage, based on sequencing and de novo assembly of an expressed gene catalog (transcriptome) from the giant horsetail (Equisetum giganteum). We demonstrate that horsetails underwent an independent paleopolyploidy during the Late Cretaceous prior to the diversification of the genus but did not experience any recent polyploidizations that could account for their high chromosome number. We also discuss the specific retention of genes following the WGD and how this may be linked to their long-term survival

Cellulose factories : advancing bioenergy production from forest trees

Fast-growing, short-rotation forest trees, such as Populus and Eucalyptus, produce large amounts of cellulose-rich biomass that could be utilized for bioenergy and biopolymer production. Major obstacles need to be overcome before the deployment of these genera as energy crops, including the effective removal of lignin and the subsequent liberation of carbohydrate constituents from wood cell walls. However, significant opportunities exist to both select for and engineer the structure and interaction of cell wall biopolymers, which could afford a means to improve processing and product development. The molecular underpinnings and regulation of cell wall carbohydrate biosynthesis are rapidly being elucidated, and are providing tools to strategically develop and guide the targeted modification required to adapt forest trees for the emerging bioeconomy. Much insight has already been gained from the perturbation of individual genes and pathways, but it is not known to what extent the natural variation in the sequence and expression of these same genes underlies the inherent variation in wood properties of field-grown trees. The integration of data from next-generation genomic technologies applied in natural and experimental populations will enable a systems genetics approach to study cell wall carbohydrate production in trees, and should advance the development of future woody bioenergy and biopolymer crops.http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1469-8137nf201