Functional investigation of five R2R3-MYB transcription factors associated with wood development in Eucalyptus using DAP-seq-ML.

A multi-tiered transcriptional network regulates xylem differentiation and secondary cell wall (SCW) formation in plants, with evidence of both conserved and lineage-specific SCW network architecture. We aimed to elucidate the roles of selected R2R3-MYB transcription factors (TFs) linked to Eucalyptus wood formation by identifying genome-wide TF binding sites and direct target genes through an improved DAP-seq protocol combined with machine learning for target gene assignment (DAP-seq-ML). We applied this to five TFs including a well-studied SCW master regulator (EgrMYB2; homolog of AtMYB83), a repressor of lignification (EgrMYB1; homolog of AtMYB4), a TF affecting SCW thickness and vessel density (EgrMYB137; homolog of PtrMYB074) and two TFs with unclear roles in SCW regulation (EgrMYB135 and EgrMYB122). Each DAP-seq TF peak set (average 12,613 peaks) was enriched for canonical R2R3-MYB binding motifs. To improve the reliability of target gene assignment to peaks, a random forest classifier was developed from Arabidopsis DAP-seq, RNA-seq, chromatin, and conserved noncoding sequence data which demonstrated significantly higher precision and recall to the baseline method of assigning genes to proximal peaks. EgrMYB1, EgrMYB2 and EgrMYB137 predicted targets showed clear enrichment for SCW-related biological processes. As validation, EgrMYB137 overexpression in transgenic Eucalyptus hairy roots increased xylem lignification, while its dominant repression in transgenic Arabidopsis and Populus reduced xylem lignification, stunted growth, and caused downregulation of SCW genes. EgrMYB137 targets overlapped significantly with those of EgrMYB2, suggesting partial functional redundancy. Our results show that DAP-seq-ML identified biologically relevant R2R3-MYB targets supported by the finding that EgrMYB137 promotes SCW lignification in planta.

Haplotype mining panel for genetic dissection and breeding in Eucalyptus.

To improve our understanding of genetic mechanisms underlying complex traits in plants, a comprehensive analysis of gene variants is required. Eucalyptus is an important forest plantation genus that is highly outbred. Trait dissection and molecular breeding in eucalypts currently relies on biallelic single-nucleotide polymorphism (SNP) markers. These markers fail to capture the large amount of haplotype diversity in these species, and thus multi-allelic markers are required. We aimed to develop a gene-based haplotype mining panel for Eucalyptus species. We generated 17 999 oligonucleotide probe sets for targeted sequencing of selected regions of 6293 genes implicated in growth and wood properties, pest and disease resistance, and abiotic stress responses. We identified and phased 195 834 SNPs using a read-based phasing approach to reveal SNP-based haplotypes. A total of 8915 target regions (at 4637 gene loci) passed tests for Mendelian inheritance. We evaluated the haplotype panel in four Eucalyptus species (E. grandis, E. urophylla, E. dunnii and E. nitens) to determine its ability to capture diversity across eucalypt species. This revealed an average of 3.13-4.52 haplotypes per target region in each species, and 33.36% of the identified haplotypes were shared by at least two species. This haplotype mining panel will enable the analysis of haplotype diversity within and between species, and provide multi-allelic markers that can be used for genome-wide association studies and gene-based breeding approaches.

Genomic consequences of artificial selection during early domestication of a wood fibre crop.

From its origins in Australia, Eucalyptus grandis has spread to every continent, except Antarctica, as a wood crop. It has been cultivated and bred for over 100 yr in places such as South Africa. Unlike most annual crops and fruit trees, domestication of E. grandis is still in its infancy, representing a unique opportunity to interrogate the genomic consequences of artificial selection early in the domestication process. To determine how a century of artificial selection has changed the genome of E. grandis, we generated single nucleotide polymorphism genotypes for 1080 individuals from three advanced South African breeding programmes using the EUChip60K chip, and investigated population structure and genome-wide differentiation patterns relative to wild progenitors. Breeding and wild populations appeared genetically distinct. We found genomic evidence of evolutionary processes known to have occurred in other plant domesticates, including interspecific introgression and intraspecific infusion from wild material. Furthermore, we found genomic regions with increased linkage disequilibrium and genetic differentiation, putatively representing early soft sweeps of selection. This is, to our knowledge, the first study of genomic signatures of domestication in a timber species looking beyond the first few generations of cultivation. Our findings highlight the importance of intra- and interspecific hybridization during early domestication.

Genome-wide association study identifies SNP markers and putative candidate genes for terpene traits important for Leptocybe invasa resistance in Eucalyptus grandis.

Terpenes are an important group of plant specialized metabolites influencing, amongst other functions, defence mechanisms against pests. We used a genome-wide association study to identify single nucleotide polymorphism (SNP) markers and putative candidate genes for terpene traits. We tested 15,387 informative SNP markers derived from genotyping 416 Eucalyptus grandis individuals for association with 3 terpene traits, 1,8-cineole, γ-terpinene, and p-cymene. A multilocus mixed model analysis identified 21 SNP markers for 1,8-cineole on chromosomes 2, 4, 6, 7, 8, 9, 10, and 11, that individually explained 3.0%-8.4% and jointly 42.7% of the phenotypic variation. Association analysis of γ-terpinene found 32 significant SNP markers on chromosomes 1, 2, 4, 5, 6, 9, and 11, explaining 3.4-15.5% and jointly 54.5% of phenotypic variation. For p-cymene, 28 significant SNP markers were identified on chromosomes 1, 2, 3, 5, 6, 7, 10, and 11, explaining 3.4-16.1% of the phenotypic variation and jointly 46.9%. Our results show that variation underlying the 3 terpene traits is influenced by a few minor loci in combination with a few major effect loci, suggesting an oligogenic nature of the traits.

A genome-wide SNP genotyping resource for tropical pine tree species.

We performed gene and genome targeted SNP discovery towards the development of a genome-wide, multispecies genotyping array for tropical pines. Pooled RNA-seq data from shoots of seedlings from five tropical pine species was used to identify transcript-based SNPs resulting in 1.3 million candidate Affymetrix SNP probe sets. In addition, we used a custom 40 K probe set to perform capture-seq in pooled DNA from 81 provenances representing the natural ranges of six tropical pine species in Mexico and Central America resulting in 563 K candidate SNP probe sets. Altogether, 300 K RNA-seq (72%) and 120 K capture-seq (28%) derived SNP probe sets were tiled on a 420 K screening array that was used to genotype 576 trees representing the 81 provenances and commercial breeding material. Based on the screening array results, 50 K SNPs were selected for commercial SNP array production including 20 K polymorphic SNPs for P. patula, P. tecunumanii, P. oocarpa and P. caribaea, 15 K for P. greggii and P. maximinoi, 13 K for P. elliottii and 8K for P. pseudostrobus. We included 9.7 K ancestry informative SNPs that will be valuable for species and hybrid discrimination. Of the 50 K SNP markers, 25% are polymorphic in only one species, while 75% are shared by two or more species. The Pitro50K SNP chip will be useful for population genomics and molecular breeding in this group of pine species that, together with their hybrids, represent the majority of fast-growing tropical and subtropical pine plantations globally.

Haplogenome assembly reveals structural variation in Eucalyptus interspecific hybrids.

BACKGROUND: De novo phased (haplo)genome assembly using long-read DNA sequencing data has improved the detection and characterization of structural variants (SVs) in plant and animal genomes. Able to span across haplotypes, long reads allow phased, haplogenome assembly in highly outbred organisms such as forest trees. Eucalyptus tree species and interspecific hybrids are the most widely planted hardwood trees with F1 hybrids of Eucalyptus grandis and E. urophylla forming the bulk of fast-growing pulpwood plantations in subtropical regions. The extent of structural variation and its effect on interspecific hybridization is unknown in these trees. As a first step towards elucidating the extent of structural variation between the genomes of E. grandis and E. urophylla, we sequenced and assembled the haplogenomes contained in an F1 hybrid of the two species. FINDINGS: Using Nanopore sequencing and a trio-binning approach, we assembled the separate haplogenomes (566.7 Mb and 544.5 Mb) to 98.0% BUSCO completion. High-density SNP genetic linkage maps of both parents allowed scaffolding of 88.0% of the haplogenome contigs into 11 pseudo-chromosomes (scaffold N50 of 43.8 Mb and 42.5 Mb for the E. grandis and E. urophylla haplogenomes, respectively). We identify 48,729 SVs between the two haplogenomes providing the first detailed insight into genome structural rearrangement in these species. The two haplogenomes have similar gene content, 35,572 and 33,915 functionally annotated genes, of which 34.7% are contained in genome rearrangements. CONCLUSIONS: Knowledge of SV and haplotype diversity in the two species will form the basis for understanding the genetic basis of hybrid superiority in these trees.

qtlXplorer: an online systems genetics browser in the Eucalyptus Genome Integrative Explorer (EucGenIE).

BACKGROUND: Affordable high-throughput DNA and RNA sequencing technologies are allowing genomic analysis of plant and animal populations and as a result empowering new systems genetics approaches to study complex traits. The availability of intuitive tools to browse and analyze the resulting large-scale genetic and genomic datasets remain a significant challenge. Furthermore, these integrative genomics approaches require innovative methods to dissect the flow and interconnectedness of biological information underlying complex trait variation. The Plant Genome Integrative Explorer (PlantGenIE.org) is a multi-species database and domain that houses online tools for model and woody plant species including Eucalyptus. Since the Eucalyptus Genome Integrative Explorer (EucGenIE) is integrated within PlantGenIE, it shares genome and expression analysis tools previously implemented within the various subdomains (ConGenIE, PopGenIE and AtGenIE). Despite the success in setting up integrative genomics databases, online tools for systems genetics modelling and high-resolution dissection of complex trait variation in plant populations have been lacking. RESULTS: We have developed qtlXplorer ( https://eucgenie.org/QTLXplorer ) for visualizing and exploring systems genetics data from genome-wide association studies including quantitative trait loci (QTLs) and expression-based QTL (eQTL) associations. This module allows users to, for example, find co-located QTLs and eQTLs using an interactive version of Circos, or explore underlying genes using JBrowse. It provides users with a means to build systems genetics models and generate hypotheses from large-scale population genomics data. We also substantially upgraded the EucGenIE resource and show how it enables users to combine genomics and systems genetics approaches to discover candidate genes involved in biotic stress responses and wood formation by focusing on two multigene families, laccases and peroxidases. CONCLUSIONS: qtlXplorer adds a new dimension, population genomics, to the EucGenIE and PlantGenIE environment. The resource will be of interest to researchers and molecular breeders working in Eucalyptus and other woody plant species. It provides an example of how systems genetics data can be integrated with functional genetics data to provide biological insight and formulate hypotheses. Importantly, integration within PlantGenIE enables novel comparative genomics analyses to be performed from population-scale data.

Genetic containment in vegetatively propagated forest trees: CRISPR disruption of LEAFY function in Eucalyptus gives sterile indeterminate inflorescences and normal juvenile development.

Eucalyptus is among the most widely planted taxa of forest trees worldwide. However, its spread as an exotic or genetically engineered form can create ecological and social problems. To mitigate gene flow via pollen and seeds, we mutated the Eucalyptus orthologue of LEAFY (LFY) by transforming a Eucalyptus grandis × urophylla wild-type hybrid and two Flowering Locus T (FT) overexpressing (and flowering) lines with CRISPR Cas9 targeting its LFY orthologue, ELFY. We achieved high rates of elfy biallelic knockouts, often approaching 100% of transgene insertion events. Frameshift mutations and deletions removing conserved amino acids caused strong floral alterations, including indeterminacy in floral development and an absence of male and female gametes. These mutants were otherwise visibly normal and did not differ statistically from transgenic controls in juvenile vegetative growth rate or leaf morphology in greenhouse trials. Genes upstream or near to ELFY in the floral development pathway were overexpressed, whereas floral organ identity genes downstream of ELFY were severely depressed. We conclude that disruption of ELFY function appears to be a useful tool for sexual containment, without causing statistically significant or large adverse effects on juvenile vegetative growth or leaf morphology.

Genomic Breeding for Diameter Growth and Tolerance to Leptocybe Gall Wasp and Botryosphaeria/Teratosphaeria Fungal Disease Complex in Eucalyptus grandis.

Eucalyptus grandis is one of the most important species for hardwood plantation forestry around the world. At present, its commercial deployment is in decline because of pests and pathogens such as Leptocybe invasa gall wasp (Lepto), and often co-occurring fungal stem diseases such as Botryosphaeria dothidea and Teratosphaeria zuluensis (BotryoTera). This study analyzed Lepto, BotryoTera, and stem diameter growth in an E. grandis multi-environmental, genetic trial. The study was established in three subtropical environments. Diameter growth and BotryoTera incidence scores were assessed on 3,334 trees, and Lepto incidence was assessed on 4,463 trees from 95 half-sib families. Using the Eucalyptus EUChip60K SNP chip, a subset of 964 trees from 93 half-sib families were genotyped with 14,347 informative SNP markers. We employed single-step genomic BLUP (ssGBLUP) to estimate genetic parameters in the genetic trial. Diameter and Lepto tolerance showed a positive genetic correlation (0.78), while BotryoTera tolerance had a negative genetic correlation with diameter growth (-0.38). The expected genetic gains for diameter growth and Lepto and BotryoTera tolerance were 12.4, 10, and -3.4%, respectively. We propose a genomic selection breeding strategy for E. grandis that addresses some of the present population structure problems.

Insect egg-induced physiological changes and transcriptional reprogramming leading to gall formation.

Gall-inducing insects and their hosts present some of the most intricate plant-herbivore interactions. Oviposition on the host is often the first cue of future herbivory and events at this early time point can affect later life stages. Many gallers are devastating plant pests, yet little information regarding the plant-insect molecular interplay exists, particularly following egg deposition. We studied the physiological and transcriptional responses of Eucalyptus following oviposition by the gall-inducing wasp, Leptocybe invasa, to explore potential mechanisms governing defence responses and gall development. RNA sequencing and microscopy were used to explore a susceptible Eucalyptus-L. invasa interaction. Infested and control material was compared over time (1-3, 7 and 90 days post oviposition) to examine the transcriptional and morphological changes. Oviposition induces accumulation of reactive oxygen species and phenolics which is reflected in the transcriptome analysis. Gene expression supports phytohormones and 10 transcription factor subfamilies as key regulators. The egg and oviposition fluid stimulate cell division resulting in gall development. Eucalyptus responses to oviposition are apparent within 24 hr. Putative defences include the oxidative burst and barrier reinforcement. However, egg and oviposition fluid stimuli may redirect these responses towards gall development.

A Genome-Wide Association Study for Resistance to the Insect Pest Leptocybe invasa in Eucalyptus grandis Reveals Genomic Regions and Positional Candidate Defense Genes.

The galling insect, Leptocybe invasa, causes significant losses in plantations of various Eucalyptus species and hybrids, threatening its economic viability. We applied a genome-wide association study (GWAS) to identify single-nucleotide polymorphism (SNP) markers associated with resistance to L. invasa. A total of 563 insect-challenged Eucalyptus grandis trees, from 61 half-sib families, were genotyped using the EUChip60K SNP chip, and we identified 15,445 informative SNP markers in the test population. Multi-locus mixed-model (MLMM) analysis identified 35 SNP markers putatively associated with resistance to L. invasa based on four discreet classes of insect damage scores: (0) not infested, (1) infested showing evidence of oviposition but no gall development, (2) infested with galls on leaves, midribs or petioles and (3) stunting and lethal gall formation. MLMM analysis identified three associated genomic regions on chromosomes 3, 7 and 8 jointly explaining 17.6% of the total phenotypic variation. SNP analysis of a validation population of 494 E. grandis trees confirmed seven SNP markers that were also detected in the initial association analysis. Based on transcriptome profiles of resistant and susceptible genotypes from an independent experiment, we identified several putative candidate genes in associated genomic loci including Nucleotide-binding ARC- domain (NB-ARC) and toll-interleukin-1-receptor-Nucleotide binding signal- Leucine rich repeat (TIR-NBS-LRR) genes. Our results suggest that Leptocybe resistance in E. grandis may be influenced by a few large-effect loci in combination with minor effect loci segregating in our test and validation populations.

Plant Biosystems Design Research Roadmap 1.0.

Human life intimately depends on plants for food, biomaterials, health, energy, and a sustainable environment. Various plants have been genetically improved mostly through breeding, along with limited modification via genetic engineering, yet they are still not able to meet the ever-increasing needs, in terms of both quantity and quality, resulting from the rapid increase in world population and expected standards of living. A step change that may address these challenges would be to expand the potential of plants using biosystems design approaches. This represents a shift in plant science research from relatively simple trial-and-error approaches to innovative strategies based on predictive models of biological systems. Plant biosystems design seeks to accelerate plant genetic improvement using genome editing and genetic circuit engineering or create novel plant systems through de novo synthesis of plant genomes. From this perspective, we present a comprehensive roadmap of plant biosystems design covering theories, principles, and technical methods, along with potential applications in basic and applied plant biology research. We highlight current challenges, future opportunities, and research priorities, along with a framework for international collaboration, towards rapid advancement of this emerging interdisciplinary area of research. Finally, we discuss the importance of social responsibility in utilizing plant biosystems design and suggest strategies for improving public perception, trust, and acceptance.

Analysis of Orthologous SECONDARY WALL-ASSOCIATED NAC DOMAIN1 (SND1) Promotor Activity in Herbaceous and Woody Angiosperms.

SECONDARY WALL-ASSOCIATED NAC DOMAIN1 (SND1) is a master regulator of fibre secondary wall deposition in Arabidopsis thaliana (Arabidopsis), with homologs in other angiosperms and gymnosperms. However, it is poorly understood to what extent the fibre-specific regulation of the SND1 promoter, and that of its orthologs, is conserved between diverged herbaceous and woody lineages. We performed a reciprocal reporter gene analysis of orthologous SND1 promoters from Arabidopsis (AthSND1), Eucalyptus grandis (EgrNAC61) and Populus alba × P. grandidentata (PagWND1A) relative to secondary cell wall-specific Cellulose Synthase4 (CesA4) and CesA7 promoters, in both a non-woody (Arabidopsis) and a woody (poplar) system. ß-glucuronidase (GUS) reporter analysis in Arabidopsis showed that the SND1 promoter was active in vascular tissues as previously reported and showed interfascicular and xylary fibre-specific expression in inflorescence stems, while reporter constructs of the woody plant-derived promoters were partial to the (pro)cambium-phloem and protoxylem. In transgenic P. tremula × P. alba plants, all three orthologous SND1 promoters expressed the GUS reporter similarly and preferentially in developing secondary xylem, ray parenchyma and cork cambium. Ours is the first study to reciprocally test orthologous SND1 promoter specificity in herbaceous and woody species, revealing diverged regulatory functions in the herbaceous system.

Dual RNA-Seq Analysis of the Pine-Fusarium circinatum Interaction in Resistant (Pinus tecunumanii) and Susceptible (Pinus patula) Hosts.

Fusarium circinatum poses a serious threat to many pine species in both commercial and natural pine forests. Knowledge regarding the molecular basis of pine-F. circinatum host-pathogen interactions could assist efforts to produce more resistant planting stock. This study aimed to identify molecular responses underlying resistance against F. circinatum. A dual RNA-seq approach was used to investigate host and pathogen expression in F. circinatum challenged Pinus tecunumanii (resistant) and Pinus patula (susceptible), at three- and seven-days post inoculation. RNA-seq reads were mapped to combined host-pathogen references for both pine species to identify differentially expressed genes (DEGs). F. circinatum genes expressed during infection showed decreased ergosterol biosynthesis in P. tecunumanii relative to P. patula. For P. tecunumanii, enriched gene ontologies and DEGs indicated roles for auxin-, ethylene-, jasmonate- and salicylate-mediated phytohormone signalling. Correspondingly, key phytohormone signaling components were down-regulated in P. patula. Key F. circinatum ergosterol biosynthesis genes were expressed at lower levels during infection of the resistant relative to the susceptible host. This study further suggests that coordination of phytohormone signaling is required for F. circinatum resistance in P. tecunumanii, while a comparatively delayed response and impaired phytohormone signaling contributes to susceptibility in P. patula.

Systems and Synthetic Biology of Forest Trees: A Bioengineering Paradigm for Woody Biomass Feedstocks.

Fast-growing forest plantations are sustainable feedstocks of plant biomass that can serve as alternatives to fossil carbon resources for materials, chemicals, and energy. Their ability to efficiently harvest light energy and carbon from the atmosphere and sequester this into metabolic precursors for lignocellulosic biopolymers and a wide range of plant specialized metabolites make them excellent biochemical production platforms and living biorefineries. Their large sizes have facilitated multi-omics analyses and systems modeling of key biological processes such as lignin biosynthesis in trees. High-throughput 'omics' approaches have also been applied in segregating tree populations where genetic variation creates abundant genetic perturbations of system components allowing construction of systems genetics models linking genes and pathways to complex trait variation. With this information in hand, it is now possible to start using synthetic biology and genome editing techniques in a bioengineering approach based on a deeper understanding and rational design of biological parts, devices, and integrated systems. However, the complexity of the biology and interacting components will require investment in big data informatics, machine learning, and intuitive visualization to fully explore multi-dimensional patterns and identify emergent properties of biological systems. Predictive systems models could be tested rapidly through high-throughput synthetic biology approaches and multigene editing. Such a bioengineering paradigm, together with accelerated genomic breeding, will be crucial for the development of a new generation of woody biorefinery crops.

Identification and functional evaluation of accessible chromatin associated with wood formation in Eucalyptus grandis.

Accessible chromatin changes dynamically during development and harbours functional regulatory regions which are poorly understood in the context of wood development. We explored the importance of accessible chromatin in Eucalyptus grandis in immature xylem generally, and MYB transcription factor-mediated transcriptional programmes specifically. We identified biologically reproducible DNase I Hypersensitive Sites (DHSs) and assessed their functional significance in immature xylem through their associations with gene expression, epigenomic data and DNA sequence conservation. We identified in vitro DNA binding sites for six secondary cell wall-associated Eucalyptus MYB (EgrMYB) transcription factors using DAP-seq, reconstructed protein-DNA networks of predicted targets based on binding sites within or outside DHSs and assessed biological enrichment of these networks with published datasets. 25 319 identified immature xylem DHSs were associated with increased transcription and significantly enriched for various epigenetic signatures (H3K4me3, H3K27me3, RNA pol II), conserved noncoding sequences and depleted single nucleotide variants. Predicted networks built from EgrMYB binding sites located in accessible chromatin were significantly enriched for systems biology datasets relevant to wood formation, whereas those occurring in inaccessible chromatin were not. Our study demonstrates that DHSs in E. grandis immature xylem, most of which are intergenic, are of functional significance to gene regulation in this tissue.

A systems genetics analysis in Eucalyptus reveals coordination of metabolic pathways associated with xylan modification in wood-forming tissues.

Acetyl- and methylglucuronic acid decorations of xylan, the dominant hemicellulose in secondary cell walls (SCWs) of woody dicots, affect its interaction with cellulose and lignin to determine SCW structure and extractability. Genes and pathways involved in these modifications may be targets for genetic engineering; however, little is known about the regulation of xylan modifications in woody plants. To address this, we assessed genetic and gene expression variation associated with xylan modification in developing xylem of Eucalyptus grandis × Eucalyptus urophylla interspecific hybrids. Expression quantitative trait locus (eQTL) mapping identified potential regulatory polymorphisms affecting gene expression modules associated with xylan modification. We identified 14 putative xylan modification genes that are members of five expression modules sharing seven trans-eQTL hotspots. The xylan modification genes are prevalent in two expression modules. The first comprises nucleotide sugar interconversion pathways supplying the essential precursors for cellulose and xylan biosynthesis. The second contains genes responsible for phenylalanine biosynthesis and S-adenosylmethionine biosynthesis required for glucuronic acid and monolignol methylation. Co-expression and co-regulation analyses also identified four metabolic sources of acetyl coenxyme A that appear to be transcriptionally coordinated with xylan modification. Our systems genetics analysis may provide new avenues for metabolic engineering to alter wood SCW biology for enhanced biomass processability.

Xylan in the Middle: Understanding Xylan Biosynthesis and Its Metabolic Dependencies Toward Improving Wood Fiber for Industrial Processing.

Lignocellulosic biomass, encompassing cellulose, lignin and hemicellulose in plant secondary cell walls (SCWs), is the most abundant source of renewable materials on earth. Currently, fast-growing woody dicots such as Eucalyptus and Populus trees are major lignocellulosic (wood fiber) feedstocks for bioproducts such as pulp, paper, cellulose, textiles, bioplastics and other biomaterials. Processing wood for these products entails separating the biomass into its three main components as efficiently as possible without compromising yield. Glucuronoxylan (xylan), the main hemicellulose present in the SCWs of hardwood trees carries chemical modifications that are associated with SCW composition and ultrastructure, and affect the recalcitrance of woody biomass to industrial processing. In this review we highlight the importance of xylan properties for industrial wood fiber processing and how gaining a greater understanding of xylan biosynthesis, specifically xylan modification, could yield novel biotechnology approaches to reduce recalcitrance or introduce novel processing traits. Altering xylan modification patterns has recently become a focus of plant SCW studies due to early findings that altered modification patterns can yield beneficial biomass processing traits. Additionally, it has been noted that plants with altered xylan composition display metabolic differences linked to changes in precursor usage. We explore the possibility of using systems biology and systems genetics approaches to gain insight into the coordination of SCW formation with other interdependent biological processes. Acetyl-CoA, s-adenosylmethionine and nucleotide sugars are precursors needed for xylan modification, however, the pathways which produce metabolic pools during different stages of fiber cell wall formation still have to be identified and their co-regulation during SCW formation elucidated. The crucial dependence on precursor metabolism provides an opportunity to alter xylan modification patterns through metabolic engineering of one or more of these interdependent pathways. The complexity of xylan biosynthesis and modification is currently a stumbling point, but it may provide new avenues for woody biomass engineering that are not possible for other biopolymers.

Organellar carbon metabolism is coordinated 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.

The plastid and mitochondrial genomes of Eucalyptus grandis.

BACKGROUND: Land plant organellar genomes have significant impact on metabolism and adaptation, and as such, accurate assembly and annotation of plant organellar genomes is an important tool in understanding the evolutionary history and interactions between these genomes. Intracellular DNA transfer is ongoing between the nuclear and organellar genomes, and can lead to significant genomic variation between, and within, species that impacts downstream analysis of genomes and transcriptomes. RESULTS: In order to facilitate further studies of cytonuclear interactions in Eucalyptus, we report an updated annotation of the E. grandis plastid genome, and the second sequenced and annotated mitochondrial genome of the Myrtales, that of E. grandis. The 478,813 bp mitochondrial genome shows the conserved protein coding regions and gene order rearrangements typical of land plants. There have been widespread insertions of organellar DNA into the E. grandis nuclear genome, which span 141 annotated nuclear genes. Further, we identify predicted editing sites to allow for the discrimination of RNA-sequencing reads between nuclear and organellar gene copies, finding that nuclear copies of organellar genes are not expressed in E. grandis. CONCLUSIONS: The implications of organellar DNA transfer to the nucleus are often ignored, despite the insight they can give into the ongoing evolution of plant genomes, and the problems they can cause in many applications of genomics. Future comparisons of the transcription and regulation of organellar genes between Eucalyptus genotypes may provide insight to the cytonuclear interactions that impact economically important traits in this widely grown lignocellulosic crop species.