Effect of Heat Treatment on Protein Self-Digestion in Ruminants' Milk.

This study investigated whether heat treatments (raw, 63 °C for 30 min, and 85 °C for 5 min) affect protein hydrolysis by endogenous enzymes in the milk of ruminants (bovine, ovine, and caprine) using a self-digestion model. Self-digestion consisted of the incubation for six hours at 37 °C of the ruminants' milk. Free amino group concentration was measured by the o-phthaldialdehyde method, and peptide sequences were identified by chromatography-mass spectrometry. Results showed that heat treatments prior to self-digestion decreased the free NH2 by 59% in bovine milk heated at 85 °C/5 min, and by 44 and 53% in caprine milk heated at 63 °C/30 min and 85 °C/5 min, respectively. However, after self-digestion, only new free amino groups were observed for the raw and heated at 63 °C/30 min milk. ß-Casein was the most cleaved protein in the raw and heated at 63 °C/30 min bovine milk. A similar trend was observed in raw ovine and caprine milk. Self-digestion increased 6.8-fold the potential antithrombin peptides in the bovine milk heated at 63 °C/30 min. Enhancing bioactive peptide abundance through self-digestion has potential applications in the industry for functional products. Overall, heat treatments affected the free amino groups according to the species and heat treatment applied, which was reflected in the varying degrees of cleaved peptide bonds and peptides released during self-digestion.

Complete Genome Sequences of Six Isolates of the Oryctes rhinoceros Nudivirus.

Oryctes rhinoceros nudivirus, a double-stranded DNA virus of the family Nudiviridae, is an important biocontrol agent of the coconut rhinoceros beetle (Coleoptera: Scarabaeidae). We present the genome sequences of six isolates of Oryctes rhinoceros nudivirus collected from the Philippines, Papua New Guinea, and Tanzania between the years 1977 and 2016.

Lysis Cassette-Mediated Exoprotein Release in Yersinia entomophaga Is Controlled by a PhoB-Like Regulator.

Secretion of exoproteins is a key component of bacterial virulence, and is tightly regulated in response to environmental stimuli and host-dependent signals. The entomopathogenic bacterium Yersinia entomophaga MH96 produces a wide range of exoproteins including its main virulence factor, the 2.46 MDa insecticidal Yen-Tc toxin complex. Previously, a high-throughput transposon-based screening assay identified the region of exoprotein release (YeRER) as essential to exoprotein release in MH96. This study defines the role of the YeRER associated ambiguous holin/endolysin-based lysis cluster (ALC) and the novel RoeA regulator in the regulation and release of exoproteins in MH96. A mutation in the ambiguous lysis cassette (ALC) region abolished exoprotein release and caused cell elongation, a phenotype able to be restored through trans-complementation with an intact ALC region. Endogenous ALC did not impact cell growth of the wild type, while artificial expression of an optimized ALC caused cell lysis. Using HolA-sfGFP and Rz1-sfGFP reporters, Rz1 expression was observed in all cells while HolA expression was limited to a small proportion of cells, which increased over time. Transcriptomic assessments found expression of the genes encoding the prominent exoproteins, including the Yen-Tc, was reduced in the roeA mutant and identified a 220 ncRNA of the YeRER intergenic region that, when trans complemented in the wildtype, abolished exoprotein release. A model for Y. entomophaga mediated exoprotein regulation and release is proposed. IMPORTANCE While theoretical models exist, there is not yet any empirical data that links ALC phage-like lysis cassettes with the release of large macro-molecular toxin complexes, such as Yen-Tc in Gram-negative bacteria. In this study, we demonstrate that the novel Y. entomophaga RoeA activates the production of exoproteins (including Yen-Tc) and the ALC at the transcriptional level. The translation of the ALC holin is confined to a subpopulation of cells that then lyse over time, indicative of a complex hierarchical regulatory network. The presence of an orthologous RoeA and a HolA like holin 5' of an eCIS Afp element in Pseudomonas chlororaphis, combined with the presented data, suggests a shared mechanism is required for the release of some large macromolecular protein assemblies, such as the Yen-Tc, and further supports classification of phage-like lysis clusters as type 10 secretion systems.

Proteomic Profile of M. longissimus thoracis from Commercial Lambs Reared in Different Forage Systems.

This study compared the protein composition of M. longissimus thoracis of lambs from six commercial forage production systems in New Zealand. A total of 286 proteins were identified based on liquid chromatography-tandem mass spectrometry. First, a binomial model showed that different production groups could be distinguished based on abundances of 16 proteins. Second, pair-wise comparisons were performed to search for protein abundance differences in meat due to animal sex (ewe vs. wether), diet (perennial ryegrass vs. chicory), and age (4 vs. 6-8 months old). Greater abundance of some myofibrillar and sarcoplasmic proteins were observed in lamb loins from ewes compared to wethers. Chicory diet and older age at slaughter were associated with meat with lower abundance of some myofibrillar proteins, possibly due to a greater proportion of muscle glycolytic fibres. The proteins that showed significant differences in their abundances due to production factors could be further investigated to understand their influence on meat quality.

Wool fiber curvature is correlated with abundance of K38 and specific keratin-associated proteins.

Curvature in mammalian fibers, such as wool and human hair, is an important feature of the functional trait of coat structure-it affects mechanical resilience and thermo-insulation. However, to examine the relationship between fiber curvature, ultrastructure and protein composition fiber diameter variability has to be minimal. To achieve this we utilised the progeny of straight-wool domestic sheep mutant rams (crimp mutants) and wild-type ewes. Proteomic and structural results of the resulting mutant/wild-type twin pairs confirmed that straight crimp mutant wool had a normal cuticle and the same cortical protein and ultrastructural building blocks as wild-type (crimpy) fibers but differed in the layout of its cortical cells and in the relative proportions of keratin (K) and keratin-associated proteins (KAPs). In the case of the crimp mutants (straight fibers), the orthocortex was distributed in a fragmented, annular ring, with some orthocortical cells near the central medulla, a pattern similar to that of straight hairs from humans and other mammals. Crimp mutant fibers were noted for the reduced abundance of some proteins in the high glycine-tyrosine class normally associated with the orthocortex, specifically the KAP6, KAP7, and KAP8 families, while proteins from the KAP16 and KAP19 were found in increased abundance. In addition to this, the type I keratin, K38, which is also associated with the orthocortex, was also found at lower abundance in the mutant fibers. Conversely, proteins from the ultra-high sulfur class normally associated with the paracortex, specifically the KAP4 and KAP9 families, were found in higher abundance.

Insights in Human Hair Curvature by Proteome Analysis of Two Distinct Hair Shapes.

Scalp hair is a universal human characteristic, and a wide range of hair shape and color variations exists. Although differences in human scalp hair shape are visually apparent, the underpinning molecular insights are yet to be fully explored. This work reports the determination of differences at the protein level between two distinct groups of hair shape: very straight samples versus very curly hair samples. An in-depth highresolution liquid-chromatography mass spectrometry proteome analysis study was performed on hair samples from 50 individuals (pooled in 10 × 5 samples) with very curly hair and 50 subjects with very straight hair (pooled in 10 × 5 samples) to decipher differences between the two experimental groups at the protein level. Our results demonstrate that a distinction between the two experimental groups (very straight vs. very curly) can be made based on their overall protein profiles in a multivariate analysis approach. Further investigation of the protein expression levels between these two groups pinpointed 13 unique proteins which were found to be significantly different between the two groups, with an adjusted p-value < 0.05 and a fold change of more than two. Although differences between the very curly and the very straight hair sample groups could be identified, linkage between population differences and curl phenotype is currently unknown and requires further investigation.

The wool proteome and fibre characteristics of three distinct genetic ovine breeds from Portugal.

Wool properties and commodity value vary considerably between breeds. In Portugal, three major ovine groups exist: Churros, Bordaleiros and Merinos. This work studies the effect of the ovine genotype on the wool proteome of such groups. Wool was collected from 15 ewes/breed and genetic groups: Churra da Terra Quente (CTQ) or Churro, Serra da Estrela (SE) or Bordaleiro and Merino Branco (MB) or Merino. Proteins were extracted and subjected to label-free proteomics analysis. A total of 50 keratinous protein groups were identified in all the samples, divided into type I and II keratins and the keratin associated proteins: high-glycine-tyrosine proteins, ultra-high sulphur proteins and high-sulphur proteins. Major differences were found between MB and CTQ with respect to K75 and K38, both medullar proteins and to a lesser extent between SE and CTQ suggesting that these might be good markers for this trait in wool. Partial least squares discriminatory analysis proved MB to be readily distinguishable from the other two breeds. Further differences were noted in keratin associated protein levels between the three breeds, normally an indicator of higher levels of orthocortex and also their relationship to high curvature, high crimp fibres like Merino. BIOLOGICAL SIGNIFICANCE: The ovine genetic type has strong effects on wool productivity parameters and quality traits. In this work, we compare the proteomes and the microscopical characteristics of the wool from three distinct ovine genetic types from Portugal: Merino, Bordaleiro and Churro. Important differences were found regarding keratin associated proteins and keratins K75 and K38, suggested as putative markers for quality traits in the wool proteome such as the average curvature.

The complete genome sequence of the nitrile biocatalyst Rhodocccus rhodochrous ATCC BAA-870.

BACKGROUND: Rhodococci are industrially important soil-dwelling Gram-positive bacteria that are well known for both nitrile hydrolysis and oxidative metabolism of aromatics. Rhodococcus rhodochrous ATCC BAA-870 is capable of metabolising a wide range of aliphatic and aromatic nitriles and amides. The genome of the organism was sequenced and analysed in order to better understand this whole cell biocatalyst. RESULTS: The genome of R. rhodochrous ATCC BAA-870 is the first Rhodococcus genome fully sequenced using Nanopore sequencing. The circular genome contains 5.9 megabase pairs (Mbp) and includes a 0.53 Mbp linear plasmid, that together encode 7548 predicted protein sequences according to BASys annotation, and 5535 predicted protein sequences according to RAST annotation. The genome contains numerous oxidoreductases, 15 identified antibiotic and secondary metabolite gene clusters, several terpene and nonribosomal peptide synthetase clusters, as well as 6 putative clusters of unknown type. The 0.53 Mbp plasmid encodes 677 predicted genes and contains the nitrile converting gene cluster, including a nitrilase, a low molecular weight nitrile hydratase, and an enantioselective amidase. Although there are fewer biotechnologically relevant enzymes compared to those found in rhodococci with larger genomes, such as the well-known Rhodococcus jostii RHA1, the abundance of transporters in combination with the myriad of enzymes found in strain BAA-870 might make it more suitable for use in industrially relevant processes than other rhodococci. CONCLUSIONS: The sequence and comprehensive description of the R. rhodochrous ATCC BAA-870 genome will facilitate the additional exploitation of rhodococci for biotechnological applications, as well as enable further characterisation of this model organism. The genome encodes a wide range of enzymes, many with unknown substrate specificities supporting potential applications in biotechnology, including nitrilases, nitrile hydratase, monooxygenases, cytochrome P450s, reductases, proteases, lipases, and transaminases.

Phylogenomic structure of Bacillus anthracis isolates in the Northern Cape Province, South Africa revealed novel single nucleotide polymorphisms.

Bacillus anthracis, the aetiological agent of anthrax, is regarded as a highly monomorphic pathogen that presents a low genetic diversity using standard molecular techniques. Whole genome sequencing and single nucleotide polymorphisms (SNPs) are definitive signatures for subtyping of B. anthracis. Here we employed whole genome single nucleotide polymorphism (wgSNP) analysis to investigate the genetic diversity of B. anthracis in the historically endemic region of Northern Cape Province (NCP), South Africa. Twenty-six isolates from anthrax outbreaks that occurred between 1998 and 2008/9 in NCP as well as from Namibia-South Africa Transfontier Conservation area and Botswana were compared to global B. anthracis genomes. Most NCP B. anthracis strains (n = 22) clustered in the A.Br.003/004 (A.Br.101) branch and are closely related to the Zimbabwe and Mozambique strains (A.Br.102 branch). A total of 4923 parsimony informative-SNPs accurately established the A.Br.003/004 phylogenetic relationships of the NCP isolates into two distinct sub-clades and SNP markers designated as A.Br.172 and A.Br.173 were developed. Other NCP strains (n = 2) grouped in the A.Br.001/002 (Sterne) branch while strains (n = 2) from the Namibia-South Africa Transfontier Conservation area and Botswana clustered in A.Br.005/006 (Ancient A) branch. The sequenced B. anthracis strains (A0094, A0096 and A0097) that clustered in the A.Br.064 (V770) clade were isolated from Vaalbos National Park and similar strains have not been isolated. The B. anthracis A0088 strain cluster with the NCP strains in the A.Br.003/004 (A.Br.172) SNP branch which has been isolated in NCP, South Africa. This study highlights the phylogenetic structure of NCP B. anthracis strains with distinctive SNP branches important for forensic tracing and novel SNP discovery purposes. The sequenced strains will serve as a means to further trace the dissemination of B. anthracis outbreaks in NCP, South Africa, and on the continent, as well as for forensic tracking on a global scale.

Scale and direction of adaptive introgression between black cottonwood (Populus trichocarpa) and balsam poplar (P. balsamifera).

Introgression can introduce novel genetic variation at a faster rate than mutation alone and result in adaptive introgression when adaptive alleles are maintained in the recipient genome over time by natural selection. A previous study from our group demonstrated adaptive introgression from Populus balsamifera into P. trichocarpa in a target genomic region. Here we expand our local ancestry analysis to the whole genome of both parents to provide a comprehensive view of introgression patterns and to identify additional candidate regions for adaptive introgression genomewide. Populus trichocarpa is a large, fast-growing tree of mild coastal regions of the Pacific Northwest, whereas P. balsamifera is a smaller stature tree of continental and boreal regions with intense winter cold. The species hybridize where they are parapatric. We detected asymmetric patterns of introgression across the whole genome of these two poplar species adapted to contrasting environments, with stronger introgression from P. balsamifera to P. trichocarpa than vice versa. Admixed P. trichocarpa individuals contained more genomic regions with unusually high levels of introgression (19 regions) and also the largest introgressed genome fragment (1.02 Mb) compared with admixed P. balsamifera (nine regions). Our analysis also revealed numerous candidate regions for adaptive introgression with strong signals of selection, notably related to disease resistance, and enriched for genes that may play crucial roles in survival and adaptation. Furthermore, we detected a potential overrepresentation of subtelomeric regions in P. balsamifera introgressed into P. trichocarpa and possible protection of sex-determining regions from interspecific gene flow.

Introgression from Populus balsamifera underlies adaptively significant variation and range boundaries in P. trichocarpa.

Introgression can be an important source of adaptive phenotypes, although conversely it can have deleterious effects. Evidence for adaptive introgression is accumulating but information on the genetic architecture of introgressed traits lags behind. Here we determine trait architecture in Populus trichocarpa under introgression from P. balsamifera using admixture mapping and phenotypic analyses. Our results reveal that admixture is a key driver of clinal adaptation and suggest that the northern range extension of P. trichocarpa depends, at least in part, on introgression from P. balsamifera. However, admixture with P. balsamifera can lead to potentially maladaptive early phenology, and a reduction in growth and disease resistance in P. trichocarpa. Strikingly, an introgressed chromosome 9 haplotype block from P. balsamifera restores the late phenology and high growth parental phenotype in admixed P. trichocarpa. This epistatic restorer block may be strongly advantageous in maximizing carbon assimilation and disease resistance in the southernmost populations where admixture has been detected. We also confirm a previously demonstrated case of adaptive introgression in chromosome 15 and show that introgression generates a transgressive chlorophyll-content phenotype. We provide strong support that introgression provides a reservoir of genetic variation associated with adaptive characters that allows improved survival in new environments.

Exploiting Natural Variation to Uncover an Alkene Biosynthetic Enzyme in Poplar.

Alkenes are linear hydrocarbons with one or more double bonds. Despite their potential as biofuels and precursors for specialty chemicals, the underlying biochemistry and genetics of alkene biosynthesis in plants remain elusive. Here, we report on a screen of natural accessions of poplar (Populus trichocarpa), revealing that the leaf cuticular waxes are predominantly composed of alkanes and alkenes. Interestingly, the accumulation of alkenes increases with leaf development, is limited to the abaxial side of the leaf, and is impaired in a few accessions. Among other genes, a ß-ketoacyl CoA synthase gene (PotriKCS1) was downregulated in leaves from non-alkene-producing accessions. We demonstrated biochemically that PotriKCS1 elongates monounsaturated fatty acids and is responsible for the recruitment of unsaturated substrates to the cuticular wax. Moreover, we found significant associations between the presence of alkenes and tree growth and resistance to leaf spot. These findings highlight the crucial role of cuticular waxes as the first point of contact with the environment, and they provide a foundation for engineering long-chain monounsaturated oils in other species.

Altering carbon allocation in hybrid poplar (Populus alba × grandidentata) impacts cell wall growth and development.

Galactinol synthase is a pivotal enzyme involved in the synthesis of the raffinose family of oligosaccharides (RFOs) that function as transport carbohydrates in the phloem, as storage compounds in sink tissues and as soluble metabolites that combat both abiotic and biotic stress in several plant species. Hybrid poplar (Populus alba × grandidentata) overexpressing the Arabidopsis thaliana GolS3 (AtGolS3) gene showed clear effects on development; the extreme overexpressing lines were stunted and had cell wall traits characteristic of tension wood, whereas lines with only moderate up-regulation grew normally and had moderately altered secondary cell wall composition and ultrastructure. Stem cross-sections of the developing xylem revealed a significant increase in the number of vessels, as well as the clear presence of a G-layer in the fibres. Furthermore, AtGolS3-OE lines possessed higher cellulose and lower lignin contents, an increase in cellulose crystallinity, and significantly altered hemicellulose-derived carbohydrates, notably manifested by their mannose and xylose contents. In addition, the transgenic plants displayed elevated xylem starch content. Transcriptome interrogation of the transgenic plants showed a significant up-regulation of genes involved in the synthesis of myo-inositol, along with genes involved in sucrose degradation. The results suggest that the overexpression of GolS and its product galactinol may serve as a molecular signal that initiates metabolic changes, culminating in a change in cell wall development and potentially the formation of tension wood.

Functional network analysis of genes differentially expressed during xylogenesis in soc1ful woody Arabidopsis plants.

Many plant genes are known to be involved in the development of cambium and wood, but how the expression and functional interaction of these genes determine the unique biology of wood remains largely unknown. We used the soc1ful loss of function mutant - the woodiest genotype known in the otherwise herbaceous model plant Arabidopsis - to investigate the expression and interactions of genes involved in secondary growth (wood formation). Detailed anatomical observations of the stem in combination with mRNA sequencing were used to assess transcriptome remodeling during xylogenesis in wild-type and woody soc1ful plants. To interpret the transcriptome changes, we constructed functional gene association networks of differentially expressed genes using the STRING database. This analysis revealed functionally enriched gene association hubs that are differentially expressed in herbaceous and woody tissues. In particular, we observed the differential expression of genes related to mechanical stress and jasmonate biosynthesis/signaling during wood formation in soc1ful plants that may be an effect of greater tension within woody tissues. Our results suggest that habit shifts from herbaceous to woody life forms observed in many angiosperm lineages could have evolved convergently by genetic changes that modulate the gene expression and interaction network, and thereby redeploy the conserved wood developmental program.

Genomic and functional approaches reveal a case of adaptive introgression from Populus balsamifera (balsam poplar) in P. trichocarpa (black cottonwood).

Natural hybrid zones in forest trees provide systems to study the transfer of adaptive genetic variation by introgression. Previous landscape genomic studies in Populus trichocarpa, a keystone tree species, indicated genomic footprints of admixture with its sister species Populus balsamifera and identified candidate genes for local adaptation. Here, we explored the patterns of introgression and signals of local adaptation in P. trichocarpa and P. balsamifera, employing genome resequencing data from three chromosomes in pure species and admixed individuals from wild populations. Local ancestry analysis in admixed P. trichocarpa revealed a telomeric region in chromosome 15 with P. balsamifera ancestry, containing several candidate genes for local adaptation. Genomic analyses revealed signals of selection in certain genes in this region (e.g. PRR5, COMT1), and functional analyses based on gene expression variation and correlations with adaptive phenotypes suggest distinct functions of the introgressed alleles. In contrast, a block of genes in chromosome 12 paralogous to the introgressed region showed no signs of introgression or signatures of selection. We hypothesize that the introgressed region in chromosome 15 has introduced modular or cassette-like variation into P. trichocarpa. These linked adaptive mutations are associated with a block of genes in chromosome 15 that appear to have undergone neo- or subfunctionalization relative to paralogs in a duplicated region on chromosome 12 that show no signatures of adaptive variation. The association between P. balsamifera introgressed alleles with the expression of adaptive traits in P. trichocarpa supports the hypothesis that this is a case of adaptive introgression in an ecologically important foundation species.

Genomic population structure and prevalence of copy number variations in South African Nguni cattle.

BACKGROUND: Copy number variations (CNVs) are modifications in DNA structure comprising of deletions, duplications, insertions and complex multi-site variants. Although CNVs are proven to be involved in a variety of phenotypic discrepancies, the full extent and consequence of CNVs is yet to be understood. To date, no such genomic characterization has been performed in indigenous South African Nguni cattle. Nguni cattle are recognized for their ability to sustain harsh environmental conditions while exhibiting enhanced resistance to disease and parasites and are thought to comprise of up to nine different ecotypes. METHODS: Illumina BovineSNP50 Beadchip data was utilized to investigate genomic population structure and the prevalence of CNVs in 492 South African Nguni cattle. PLINK, ADMIXTURE, R, gPLINK and Haploview software was utilized for quality control, population structure and haplotype block determination. PennCNV hidden Markov model identified CNVs and genes contained within and 10 Mb downstream from reported CNVs. PANTHER and Ensembl databases were subsequently utilized for gene annotation analyses. RESULTS: Population structure analyses on Nguni cattle revealed 5 sub-populations with a possible sub-structure evident at K equal to 8. Four hundred and thirty three CNVs that formed 334 CNVRs ranging from 30 kb to 1 Mb in size are reported. Only 231 of the 492 animals demonstrated CNVRs. Two hundred and eighty nine genes were observed within CNVRs identified. Of these 149, 28, 44, 2 and 14 genes were unique to sub-populations A, B, C, D and E respectively. Gene ontology analyses demonstrated a number of pathways to be represented by respective genes, including immune response, response to abiotic stress and biological regulation processess. CONCLUSIONS: CNVs may explain part of the phenotypic diversity and the enhanced adaptation evident in Nguni cattle. Genes involved in a number of cellular components, biological processes and molecular functions are reported within CNVRs identified. The significance of such CNVRs and the possible effect thereof needs to be ascertained and may hold interesting insight into the functional and adaptive consequence of CNVs in cattle.

The transcriptional landscape of insect galls: psyllid (Hemiptera) gall formation in Hawaiian Metrosideros polymorpha (Myrtaceae).

BACKGROUND: Recent studies show that galling Hymenoptera and Diptera are able to synthesize the plant hormone indole-3-acetic acid (auxin) from tryptophan and that plant response to insect-produced auxin is implicated in gall formation. We examined the leaf transcriptome of galled and ungalled leaves of individuals of the Hawaiian endemic plant Metrosideros polymorpha (Myrtaceae) subject to infestation by psyllid (Hemiptera) gall-makers in the genus Trioza (Triozidae). RESULTS: Transcript libraries were sequenced using Illumina technology and the reads assembled de novo into contigs. Functional identification of contigs followed a two-step procedure, first identifying contigs by comparison to the completely sequenced genome of the related Eucalyptus, followed by identifying the equivalent Arabidopsis gene using a pre-computed mapping between Eucalyptus and Arabidopsis genes. This allowed us to use the rich functional annotation of the Arabidopsis genome to assess the transcriptional landscape of galling in Metrosideros. Comparing galled and ungalled leaves, we find a highly significant enrichment of expressed genes with a gene ontology (GO) annotation to auxin response in the former. One gene consistently expressed in all galled trees examined but not detected in any libraries from ungalled leaves was the Metrosideros version of SMALL AUXIN UPREGULATED (SAUR) 67 which appears to be a marker for leaf-galling in Metrosideros. CONCLUSIONS: We conclude that an auxin response is involved in galling by Metrosideros psyllids. The possibility should therefore be considered that psyllids (like other insects examined) are able to synthesize auxin.

The Eucalyptus terpene synthase gene family.

BACKGROUND: Terpenoids are abundant in the foliage of Eucalyptus, providing the characteristic smell as well as being valuable economically and influencing ecological interactions. Quantitative and qualitative inter- and intra- specific variation of terpenes is common in eucalypts. RESULTS: The genome sequences of Eucalyptus grandis and E. globulus were mined for terpene synthase genes (TPS) and compared to other plant species. We investigated the relative expression of TPS in seven plant tissues and functionally characterized five TPS genes from E. grandis. Compared to other sequenced plant genomes, Eucalyptus grandis has the largest number of putative functional TPS genes of any sequenced plant. We discovered 113 and 106 putative functional TPS genes in E. grandis and E. globulus, respectively. All but one TPS from E. grandis were expressed in at least one of seven plant tissues examined. Genomic clusters of up to 20 genes were identified. Many TPS are expressed in tissues other than leaves which invites a re-evaluation of the function of terpenes in Eucalyptus. CONCLUSIONS: Our data indicate that terpenes in Eucalyptus may play a wider role in biotic and abiotic interactions than previously thought. Tissue specific expression is common and the possibility of stress induction needs further investigation. Phylogenetic comparison of the two investigated Eucalyptus species gives insight about recent evolution of different clades within the TPS gene family. While the majority of TPS genes occur in orthologous pairs some clades show evidence of recent gene duplication, as well as loss of function.

Comparative analysis of plant carbohydrate active enZymes and their role in xylogenesis.

BACKGROUND: Carbohydrate metabolism is a key feature of vascular plant architecture, and is of particular importance in large woody species, where lignocellulosic biomass is responsible for bearing the bulk of the stem and crown. Since Carbohydrate Active enZymes (CAZymes) in plants are responsible for the synthesis, modification and degradation of carbohydrate biopolymers, the differences in gene copy number and regulation between woody and herbaceous species have been highlighted previously. There are still many unanswered questions about the role of CAZymes in land plant evolution and the formation of wood, a strong carbohydrate sink. RESULTS: Here, twenty-two publically available plant genomes were used to characterize the frequency, diversity and complexity of CAZymes in plants. We find that a conserved suite of CAZymes is a feature of land plant evolution, with similar diversity and complexity regardless of growth habit and form. In addition, we compared the diversity and levels of CAZyme gene expression during wood formation in trees using mRNA-seq data from two distantly related angiosperm tree species Eucalyptus grandis and Populus trichocarpa, highlighting the major CAZyme classes involved in xylogenesis and lignocellulosic biomass production. CONCLUSIONS: CAZyme domain ratio across embryophytes is maintained, and the diversity of CAZyme domains is similar in all land plants, regardless of woody habit. The stoichiometric conservation of gene expression in woody and non-woody tissues of Eucalyptus and Populus are indicative of gene balance preservation.

Genome-wide analysis of the lignin toolbox of Eucalyptus grandis.

Lignin, a major component of secondary cell walls, hinders the optimal processing of wood for industrial uses. The recent availability of the Eucalyptus grandis genome sequence allows comprehensive analysis of the genes encoding the 11 protein families specific to the lignin branch of the phenylpropanoid pathway and identification of those mainly involved in xylem developmental lignification. We performed genome-wide identification of putative members of the lignin gene families, followed by comparative phylogenetic studies focusing on bona fide clades inferred from genes functionally characterized in other species. RNA-seq and microfluid real-time quantitative PCR (RT-qPCR) expression data were used to investigate the developmental and environmental responsive expression patterns of the genes. The phylogenetic analysis revealed that 38 E. grandis genes are located in bona fide lignification clades. Four multigene families (shikimate O-hydroxycinnamoyltransferase (HCT), p-coumarate 3-hydroxylase (C3H), caffeate/5-hydroxyferulate O-methyltransferase (COMT) and phenylalanine ammonia-lyase (PAL)) are expanded by tandem gene duplication compared with other plant species. Seventeen of the 38 genes exhibited strong, preferential expression in highly lignified tissues, probably representing the E. grandis core lignification toolbox. The identification of major genes involved in lignin biosynthesis in E. grandis, the most widely planted hardwood crop world-wide, provides the foundation for the development of biotechnology approaches to develop tree varieties with enhanced processing qualities.