Eshchar Mizrachi

The genome of Eucalyptus grandis

Eucalypts are the world’s most widely planted hardwood trees. Their outstanding diversity, adaptability and growth have made them a global renewable resource of fibre and energy. We sequenced and assembled >94% of the 640-megabase genome of Eucalyptus grandis. Of 36,376 predicted protein-coding genes, 34% occur in tandem duplications, the largest proportion thus far in plant genomes. Eucalyptus also shows the highest diversity of genes for specialized metabolites such as terpenes that act as chemical defence and provide unique pharmaceutical oils. Genome sequencing of the E. grandis sister species E. globulus and a set of inbred E. grandis tree genomes reveals dynamic genome evolution and hotspots of inbreeding depression. The E. grandis genome is the first reference for the eudicot order Myrtales and is placed here sister to the eurosids. This resource expands our understanding of the unique biology of large woody perennials and provides a powerful tool to accelerate comparative biology, breeding and biotechnology.

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

BackgroundDe novo 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 Eucalyptus tree species (E. grandis) 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.ResultsWe present an extensive expressed gene catalog for a commercially grown E. grandis × E. urophylla hybrid clone constructed using only Illumina mRNA-Seq technology and de novo 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 Eucalyptus 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.ConclusionsDe novo assembly of Illumina mRNA-Seq reads is an efficient approach for transcriptome sequencing and profiling in Eucalyptus and other non-model organisms. The transcriptome resource (Eucspresso, http://eucspresso.bi.up.ac.za/) generated by this study will be of value for genomic analysis of woody biomass production in Eucalyptus and for comparative genomic analysis of growth and development in woody and herbaceous plants.

SND2, a NAC transcription factor gene, regulates genes involved in secondary cell wall development in Arabidopsis fibres and increases fibre cell area in Eucalyptus

BackgroundNAC domain transcription factors initiate secondary cell wall biosynthesis in Arabidopsis fibres and vessels by activating numerous transcriptional regulators and biosynthetic genes. NAC family member SND2 is an indirect target of a principal regulator of fibre secondary cell wall formation, SND1. A previous study showed that overexpression of SND2 produced a fibre cell-specific increase in secondary cell wall thickness in Arabidopsis stems, and that the protein was able to transactivate the cellulose synthase8 (CesA8) promoter. However, the full repertoire of genes regulated by SND2 is unknown, and the effect of its overexpression on cell wall chemistry remains unexplored.ResultsWe overexpressed SND2 in Arabidopsis and analyzed homozygous lines with regards to stem chemistry, biomass and fibre secondary cell wall thickness. A line showing upregulation of CesA8 was selected for transcriptome-wide gene expression profiling. We found evidence for upregulation of biosynthetic genes associated with cellulose, xylan, mannan and lignin polymerization in this line, in agreement with significant co-expression of these genes with native SND2 transcripts according to public microarray repositories. Only minor alterations in cell wall chemistry were detected. Transcription factor MYB103, in addition to SND1, was upregulated in SND2-overexpressing plants, and we detected upregulation of genes encoding components of a signal transduction machinery recently proposed to initiate secondary cell wall formation. Several homozygous T4 and hemizygous T1 transgenic lines with pronounced SND2 overexpression levels revealed a negative impact on fibre wall deposition, which may be indirectly attributable to excessive overexpression rather than co-suppression. Conversely, overexpression of SND2 in Eucalyptus stems led to increased fibre cross-sectional cell area.ConclusionsThis study supports a function for SND2 in the regulation of cellulose and hemicellulose biosynthetic genes in addition of those involved in lignin polymerization and signalling. SND2 seems to occupy a subordinate but central tier in the secondary cell wall transcriptional network. Our results reveal phenotypic differences in the effect of SND2 overexpression between woody and herbaceous stems and emphasize the importance of expression thresholds in transcription factor studies.

Navigating the transcriptional roadmap regulating plant secondary cell wall deposition

The current status of lignocellulosic biomass as an invaluable resource in industry, agriculture, and health has spurred increased interest in understanding the transcriptional regulation of secondary cell wall (SCW) biosynthesis. The last decade of research has revealed an extensive network of NAC, MYB and other families of transcription factors regulating Arabidopsis SCW biosynthesis, and numerous studies have explored SCW-related transcription factors in other dicots and monocots. Whilst the general structure of the Arabidopsis network has been a topic of several reviews, they have not comprehensively represented the detailed protein–DNA and protein–protein interactions described in the literature, and an understanding of network dynamics and functionality has not yet been achieved for SCW formation. Furthermore the methodologies employed in studies of SCW transcriptional regulation have not received much attention, especially in the case of non-model organisms. In this review, we have reconstructed the most exhaustive literature-based network representations to date of SCW transcriptional regulation in Arabidopsis. We include a manipulable Cytoscape representation of the Arabidopsis SCW transcriptional network to aid in future studies, along with a list of supporting literature for each documented interaction. Amongst other topics, we discuss the various components of the network, its evolutionary conservation in plants, putative modules and dynamic mechanisms that may influence network function, and the approaches that have been employed in network inference. Future research should aim to better understand network function and its response to dynamic perturbations, whilst the development and application of genome-wide approaches such as ChIP-seq and systems genetics are in progress for the study of SCW transcriptional regulation in non-model organisms.

Horsetails Are Ancient Polyploids: Evidence from Equisetum giganteum

An ancient polypoloidy event in horsetails during the late Cretaceous predates the divergence of todays extant species and may have contributed to the longevity of the genus Equisetum. 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.

The Eucalyptus grandis Genome Project: Genome and transcriptome resources for comparative analysis of woody plant biology

Background The International Year of Forests 2011 [http://www. un.org/en/events/iyof2011/] will be a milestone for forest tree genomics. The draft genome sequence of Eucalyptus grandis was released in January 2011 in the USA (Phytozome [http://www.phytozome.net]) and in Belgium (BOGAS, [http://bioinformatics.psb.ugent.be/webtools/bogas/]). The genome sequencing was funded by the US Department of Energy (DOE) and performed at the DOE Joint Genome Institute (JGI) in collaboration with members of the Eucalyptus Genome Network (EUCAGEN, [http://www.eucagen.org]) who contributed genetic materials, linkage maps, EST resources and bioinformatics support. The E. grandis genome together with that of Populus trichocarpa[1]and other woody plant genomes recently completed (e.g. Vitis, Cacao, Prunus, Citrus and Malus)will provide excellent opportunities for comparative studies of the unique biology of woody plants. Eucalypts are currently the most widely grown hardwood fibre crop in the world and eucalypt breeding programs will benefit greatly from the new genomic resources. The reference genome sequence of Eucalyptus, a foundation tree genus in Australia comprising more than 70% of the native forest estate, will also offer important benefits for ecological and evolutionary biology studies. We report the sequencing, assembly and annotation of the E. grandis genome. Genome sequencing and assembly Whole-genome (8X) shotgun sequencing was performed for a partially inbred (S1), 17-year-old tree of E. grandis (est. genome size 640 Mbp, n = 11), BRASUZ1 (Suzano, Brazil). A total of 7.7 million Sanger reads (5.4 Gbp) were produced from plasmid, fosmid and BAC libraries. An inbred genotype was selected to circumvent perceived problems with the assembly of a highly heterozygous eucalypt genome. However, microsatellite genotyping showed that BRASUZ1 was much less homozygous than expected, with large parts of the genome remaining heterozygous presumably due to viability selection. This finding was confirmed during the assembly of the S1 genome approximately 25% of the assembly occurred in two haplotypes of 3-4X coverage, while the remainder of the genome assembled into a single haplotype of 6-7X coverage. Linkage maps with over 2400 DArT and microsatellite markers were subsequently used as a framework for the assembly of 11 large chromosome scaffolds. The chromosome scaffolds contained 88% (605 Mbp) of the draft assembly, with the remainder of the assembly sequence (85 Mbp) in 4941 smaller scaffolds. Based on similarity searches with 1.6 million ESTs from BRASUZ1, it was estimated that 96% of expressed gene loci were included in the 11 chromosome assemblies.

Comparative interrogation of the developing xylem transcriptomes of two wood‐forming species: Populus trichocarpa and Eucalyptus grandis

Wood formation is a complex developmental process governed by genetic and environmental stimuli. Populus and Eucalyptus are fast-growing, high-yielding tree genera that represent ecologically and economically important species suitable for generating significant lignocellulosic biomass. Comparative analysis of the developing xylem and leaf transcriptomes of Populus trichocarpa and Eucalyptus grandis together with phylogenetic analyses identified clusters of homologous genes preferentially expressed during xylem formation in both species. A conserved set of 336 single gene pairs showed highly similar xylem preferential expression patterns, as well as evidence of high functional constraint. Individual members of multi-gene orthologous clusters known to be involved in secondary cell wall biosynthesis also showed conserved xylem expression profiles. However, species-specific expression as well as opposite (xylem versus leaf) expression patterns observed for a subset of genes suggest subtle differences in the transcriptional regulation important for xylem development in each species. Using sequence similarity and gene expression status, we identified functional homologs likely to be involved in xylem developmental and biosynthetic processes in Populus and Eucalyptus. Our study suggests that, while genes involved in secondary cell wall biosynthesis show high levels of gene expression conservation, differential regulation of some xylem development genes may give rise to unique xylem properties.

Cell Wall-related Proteins of Unknown Function: Missing Links in Plant Cell Wall Development

Lignocellulosic biomass is an important feedstock for the pulp and paper industry as well as emerging biofuel and biomaterial industries. However, the recalcitrance of the secondary cell wall to chemical or enzymatic degradation remains a major hurdle for efficient extraction of economically important biopolymers such as cellulose. It has been estimated that approximately 10-15% of about 27,000 protein-coding genes in the Arabidopsis genome are dedicated to cell wall development; however, only about 130 Arabidopsis genes thus far have experimental evidence validating cell wall function. While many genes have been implicated through co-expression analysis with known genes, a large number are broadly classified as proteins of unknown function (PUFs). Recently the functionality of some of these unknown proteins in cell wall development has been revealed using reverse genetic approaches. Given the large number of cell wall-related PUFs, how do we approach and subsequently prioritize the investigation of such unknown genes that may be essential to or influence plant cell wall development and structure? Here, we address the aforementioned question in two parts; we first identify the different kinds of PUFs based on known and predicted features such as protein domains. Knowledge of inherent features of PUFs may allow for functional inference and a concomitant link to biological context. Secondly, we discuss omics-based technologies and approaches that are helping identify and prioritize cell wall-related PUFs by functional association. In this way, hypothesis-driven experiments can be designed for functional elucidation of many proteins that remain missing links in our understanding of plant cell wall biosynthesis.

Recombinant hyperthermophilic enzyme expression in plants: a novel approach for lignocellulose digestion

Plant biomass, as an abundant renewable carbon source, is a promising alternative to fossil fuels. However, the enzymes most commonly used for depolymerization of lignocellulosic biomass are expensive, and the development of cost-effective alternative conversion technologies would be desirable. One possible option is the heterologous expression of genes encoding lignocellulose-digesting enzymes in plant tissues. To overcome simultaneously issues of toxicity and incompatibility with high-temperature steam explosion processes, the use of heterologous genes encoding hyperthermophilic enzymes may be an attractive alternative. This approach could reduce the need for exogenous enzyme additions prior to fermentation, reducing the cost of the complete processing operation. This review highlights recent advances and future prospects for using hyperthermophilic enzymes in the biofuels industry.

The Eucalyptus genome integrative explorer (EucGenIE): a resource for Eucalyptus genomics and transcriptomics

Background The fast growth and good wood properties of Eucalyptus tree species and hybrids make them excellent renewable sources of fiber for pulp and paper production, and woody biomass for bioenergy production. Our research is aimed at understanding the genetic regulation of wood formation in eucalypts, with a focus on transcriptomes, regulatory sequences and gene families involved in secondary cell wall biosynthesis.