Range-wide differential adaptation and genomic offset in critically endangered Asian rosewoods.

In the billion-dollar global illegal wildlife trade, rosewoods have been the world's most trafficked wild product since 2005. Dalbergia cochinchinensis and Dalbergia oliveri are the most sought-after rosewoods in the Greater Mekong Subregion. They are exposed to significant genetic risks and the lack of knowledge on their adaptability limits the effectiveness of conservation efforts. Here, we present genome assemblies and range-wide genomic scans of adaptive variation, together with predictions of genomic offset to climate change. Adaptive genomic variation was differentially associated with temperature and precipitation-related variables between the species, although their natural ranges overlap. The findings are consistent with differences in pioneering ability and in drought tolerance. We predict their genomic offsets will increase over time and with increasing carbon emission pathway but at a faster pace in D. cochinchinensis than in D. oliveri. These results and the distinct gene-environment association in the eastern coastal edge of Vietnam suggest species-specific conservation actions: germplasm representation across the range in D. cochinchinensis and focused on hotspots of genomic offset in D. oliveri. We translated our genomic models into a seed source matching application, seedeR, to rapidly inform restoration efforts. Our ecological genomic research uncovering contrasting selection forces acting in sympatric rosewoods is of relevance to conserving tropical trees globally and combating risks from climate change.

Long-insert sequence capture detects high copy numbers in a defence-related beta-glucosidase gene ßglu-1 with large variations in white spruce but not Norway spruce.

Conifers are long-lived and slow-evolving, thus requiring effective defences against their fast-evolving insect natural enemies. The copy number variation (CNV) of two key acetophenone biosynthesis genes Ugt5/Ugt5b and ßglu-1 may provide a plausible mechanism underlying the constitutively variable defence in white spruce (Picea glauca) against its primary defoliator, spruce budworm. This study develops a long-insert sequence capture probe set (Picea_hung_p1.0) for quantifying copy number of ßglu-1-like, Ugt5-like genes and single-copy genes on 38 Norway spruce (Picea abies) and 40 P. glauca individuals from eight and nine provenances across Europe and North America respectively. We developed local assemblies (Piabi_c1.0 and Pigla_c.1.0), full-length transcriptomes (PIAB_v1 and PIGL_v1), and gene models to characterise the diversity of ßglu-1 and Ugt5 genes. We observed very large copy numbers of ßglu-1, with up to 381 copies in a single P. glauca individual. We observed among-provenance CNV of ßglu-1 in P. glauca but not P. abies. Ugt5b was predominantly single-copy in both species. This study generates critical hypotheses for testing the emergence and mechanism of extreme CNV, the dosage effect on phenotype, and the varying copy number of genes with the same pathway. We demonstrate new approaches to overcome experimental challenges in genomic research in conifer defences.

Spruce giga-genomes: structurally similar yet distinctive with differentially expanding gene families and rapidly evolving genes.

Spruces (Picea spp.) are coniferous trees widespread in boreal and mountainous forests of the northern hemisphere, with large economic significance and enormous contributions to global carbon sequestration. Spruces harbor very large genomes with high repetitiveness, hampering their comparative analysis. Here, we present and compare the genomes of four different North American spruces: the genome assemblies for Engelmann spruce (Picea engelmannii) and Sitka spruce (Picea sitchensis) together with improved and more contiguous genome assemblies for white spruce (Picea glauca) and for a naturally occurring introgress of these three species known as interior spruce (P. engelmannii × glauca × sitchensis). The genomes were structurally similar, and a large part of scaffolds could be anchored to a genetic map. The composition of the interior spruce genome indicated asymmetric contributions from the three ancestral genomes. Phylogenetic analysis of the nuclear and organelle genomes revealed a topology indicative of ancient reticulation. Different patterns of expansion of gene families among genomes were observed and related with presumed diversifying ecological adaptations. We identified rapidly evolving genes that harbored high rates of non-synonymous polymorphisms relative to synonymous ones, indicative of positive selection and its hitchhiking effects. These gene sets were mostly distinct between the genomes of ecologically contrasted species, and signatures of convergent balancing selection were detected. Stress and stimulus response was identified as the most frequent function assigned to expanding gene families and rapidly evolving genes. These two aspects of genomic evolution were complementary in their contribution to divergent evolution of presumed adaptive nature. These more contiguous spruce giga-genome sequences should strengthen our understanding of conifer genome structure and evolution, as their comparison offers clues into the genetic basis of adaptation and ecology of conifers at the genomic level. They will also provide tools to better monitor natural genetic diversity and improve the management of conifer forests. The genomes of four closely related North American spruces indicate that their high similarity at the morphological level is paralleled by the high conservation of their physical genome structure. Yet, the evidence of divergent evolution is apparent in their rapidly evolving genomes, supported by differential expansion of key gene families and large sets of genes under positive selection, largely in relation to stimulus and environmental stress response.

Comparative transcriptomic responses of European and Japanese larches to infection by Phytophthora ramorum.

BACKGROUND AND OBJECTIVES: Phytophthora ramorum severely affects both European larch (EL) and Japanese larch (JL) trees as indicated by high levels of mortality particularly in the UK. Field observations suggested that EL is less severely affected and so may be less susceptible to P. ramorum than JL; however, controlled inoculations have produced inconsistent or non-statistically significant differences. The present study aimed to compare RNA transcript accumulation profiles in EL and JL in response to inoculation with P. ramorum to improve our understanding of their defence responses. METHODOLOGY: RNA-sequencing was carried out on bark tissues following the inoculation with P. ramorum of potted saplings in both EL and JL carried out under controlled environment conditions, with sampling at 1, 3, 10, and 25 days post inoculation in infected and control plants. RESULTS: All of the inoculated trees rapidly developed lesions but no statistically significant differences were found in lesion lengths between EL and JL. RNA-Sequencing comparing control and inoculate saplings identified key differences in differentially expressed genes (DEGs) between the two larch species. European larch had rapid induction of defence genes within 24 hours of infection followed by sustained expression until 25 days after inoculation. Results in JL were more varied; upregulation was stronger but more transient and represented fewer defence pathways. Gene enrichment analyses highlighted differences in jasmonate signalling and regulation including NPR1 upregulation in EL only, and specific aspects of secondary metabolism. Some DEGs were represented by multiple responsive copies including lipoxygenase, chalcone synthase and nucleotide-binding, leucine-rich-repeat genes. CONCLUSION: The variations between EL and JL in responsive DEGs of interest as potentially related to differences seen in the field and should be considered in the selection of trees for planting and future breeding.

Association genetics of acetophenone defence against spruce budworm in mature white spruce.

BACKGROUND: Outbreaks of spruce budworm (SBW, Choristoneura fumiferana Clem.) cause major recurrent damage in boreal conifers such as white spruce (Picea glauca [Moench] Voss) and large losses of forest biomass in North America. Although defensive phenolic compounds have recently been linked to chemical resistance against SBW, their genetic basis remains poorly understood in forest trees, especially in conifers. Here, we used diverse association genetics approaches to discover genes and their variants that may control the accumulation of acetophenones, and dissect the genetic architecture of these defence compounds against SBW in white spruce mature trees. RESULTS: Out of 4747 single nucleotide polymorphisms (SNPs) from 2312 genes genotyped in a population of 211 unrelated individuals, genetic association analyses identified 35 SNPs in 33 different genes that were significantly associated with the defence traits by using single-locus, multi-locus and multi-trait approaches. The multi-locus approach was particularly effective at detecting SNP-trait associations that explained a large fraction of the phenotypic variance (from 20 to 43%). Significant genes were regulatory including the NAC transcription factor, or they were involved in carbohydrate metabolism, falling into the binding, catalytic or transporter activity functional classes. Most of them were highly expressed in foliage. Weak positive phenotypic correlations were observed between defence and growth traits, indicating little or no evidence of defence-growth trade-offs. CONCLUSIONS: This study provides new insights on the genetic architecture of tree defence traits, contributing to our understanding of the physiology of resistance mechanisms to biotic factors and providing a basis for the genetic improvement of the constitutive defence of white spruce against SBW.

Evolution of the biosynthesis of two hydroxyacetophenones in plants.

Acetophenones are phenolic metabolites of plant species. A metabolic route for the biosynthesis and release of 2 defence-related hydroxyacetophenones in white spruce (Picea glauca) was recently proposed to involve 3 phases: (a) biosynthesis of the acetophenone aglycons catalysed by a currently unknown set of enzymes, (b) formation and accumulation of the corresponding glycosides catalysed by a glucosyltransferase, and (c) release of the aglycons catalysed by a glucosylhydrolase (PgßGLU-1). We tested if this biosynthetic model is conserved across Pinaceae and land plant species. We assayed and surveyed the literature and sequence databases for possible patterns of the presence of the acetophenone aglycons piceol and pungenol and their glucosides, as well as sequences and expression of Pgßglu-1 orthologues. In the Pinaceae, the 3 phases of the biosynthetic model are present and differences in expression of Pgßglu-1 gene orthologues explain some of the interspecific variation in hydroxyacetophenones. The phylogenetic signal in the metabolite phenotypes was low across species of 6 plant divisions. Putative orthologues of PgßGLU-1 do not form a monophyletic group in species producing hydroxyacetophenones. The biosynthetic model for acetophenones appears to be conserved across Pinaceae, whereas convergent evolution has led to the production of acetophenone glucosides across land plants.

Expression of the ß-glucosidase gene Pgßglu-1 underpins natural resistance of white spruce against spruce budworm.

Periodic outbreaks of spruce budworm (SBW) affect large areas of ecologically and economically important conifer forests in North America, causing tree mortality and reduced forest productivity. Host resistance against SBW has been linked to growth phenology and the chemical composition of foliage, but the underlying molecular mechanisms and population variation are largely unknown. Using a genomics approach, we discovered a ß-glucosidase gene, Pgßglu-1, whose expression levels and function underpin natural resistance to SBW in mature white spruce (Picea glauca) trees. In phenotypically resistant trees, Pgßglu-1 transcripts were up to 1000 times more abundant than in non-resistant trees and were highly enriched in foliage. The encoded PgßGLU-1 enzyme catalysed the cleavage of acetophenone sugar conjugates to release the aglycons piceol and pungenol. These aglycons were previously shown to be active against SBW. Levels of Pgßglu-1 transcripts and biologically active acetophenone aglycons were substantially different between resistant and non-resistant trees over time, were positively correlated with each other and were highly variable in a natural white spruce population. These results suggest that expression of Pgßglu-1 and accumulation of acetophenone aglycons is a constitutive defence mechanism in white spruce. The progeny of resistant trees had higher Pgßglu-1 gene expression than non-resistant progeny, indicating that the trait is heritable. With reported increases in the intensity of SBW outbreaks, influenced by climate, variation of Pgßglu-1 transcript expression, PgßGLU-1 enzyme activity and acetophenone accumulation may serve as resistance markers to better predict impacts of SBW in both managed and wild spruce populations.

Conifer genomics and adaptation: at the crossroads of genetic diversity and genome function.

Conifers have been understudied at the genomic level despite their worldwide ecological and economic importance but the situation is rapidly changing with the development of next generation sequencing (NGS) technologies. With NGS, genomics research has simultaneously gained in speed, magnitude and scope. In just a few years, genomes of 20-24 gigabases have been sequenced for several conifers, with several others expected in the near future. Biological insights have resulted from recent sequencing initiatives as well as genetic mapping, gene expression profiling and gene discovery research over nearly two decades. We review the knowledge arising from conifer genomics research emphasizing genome evolution and the genomic basis of adaptation, and outline emerging questions and knowledge gaps. We discuss future directions in three areas with potential inputs from NGS technologies: the evolutionary impacts of adaptation in conifers based on the adaptation-by-speciation model; the contributions of genetic variability of gene expression in adaptation; and the development of a broader understanding of genetic diversity and its impacts on genome function. These research directions promise to sustain research aimed at addressing the emerging challenges of adaptation that face conifer trees.

Modular organization of the white spruce (Picea glauca) transcriptome reveals functional organization and evolutionary signatures.

Transcript profiling has shown the molecular bases of several biological processes in plants but few studies have developed an understanding of overall transcriptome variation. We investigated transcriptome structure in white spruce (Picea glauca), aiming to delineate its modular organization and associated functional and evolutionary attributes. Microarray analyses were used to: identify and functionally characterize groups of co-expressed genes; investigate expressional and functional diversity of vascular tissue preferential genes which were conserved among Picea species, and identify expression networks underlying wood formation. We classified 22 857 genes as variable (79%; 22 coexpression groups) or invariant (21%) by profiling across several vegetative tissues. Modular organization and complex transcriptome restructuring among vascular tissue preferential genes was revealed by their assignment to coexpression groups with partially overlapping profiles and partially distinct functions. Integrated analyses of tissue-based and temporally variable profiles identified secondary xylem gene networks, showed their remodelling over a growing season and identified PgNAC-7 (no apical meristerm (NAM), Arabidopsis transcription activation factor (ATAF) and cup-shaped cotyledon (CUC) transcription factor 007 in Picea glauca) as a major hub gene specific to earlywood formation. Reference profiling identified comprehensive, statistically robust coexpressed groups, revealing that modular organization underpins the evolutionary conservation of the transcriptome structure.

Prepublication data sharing.

Rapid release of prepublication data has served the field of genomics well. Attendees at a workshop in Toronto recommend extending the practice to other biological data sets.

Opposite action of R2R3-MYBs from different subgroups on key genes of the shikimate and monolignol pathways in spruce.

Redundancy and competition between R2R3-MYB activators and repressors on common target genes has been proposed as a fine-tuning mechanism for the regulation of plant secondary metabolism. This hypothesis was tested in white spruce [Picea glauca (Moench) Voss] by investigating the effects of R2R3-MYBs from different subgroups on common targets from distinct metabolic pathways. Comparative analysis of transcript profiling data in spruces overexpressing R2R3-MYBs from loblolly pine (Pinus taeda L.), PtMYB1, PtMYB8, and PtMYB14, defined a set of common genes that display opposite regulation effects. The relationship between the closest MYB homologues and 33 putative target genes was explored by quantitative PCR expression profiling in wild-type P. glauca plants during the diurnal cycle. Significant Spearman's correlation estimates were consistent with the proposed opposite effect of different R2R3-MYBs on several putative target genes in a time-related and tissue-preferential manner. Expression of sequences coding for 4CL, DHS2, COMT1, SHM4, and a lipase thio/esterase positively correlated with that of PgMYB1 and PgMYB8, but negatively with that of PgMYB14 and PgMYB15. Complementary electrophoretic mobility shift assay (EMSA) and transactivation assay provided experimental evidence that these different R2R3-MYBs are able to bind similar AC cis-elements in the promoter region of Pg4CL and PgDHS2 genes but have opposite effects on their expression. Competitive binding EMSA experiments showed that PgMYB8 competes more strongly than PgMYB15 for the AC-I MYB binding site in the Pg4CL promoter. Together, the results bring a new perspective to the action of R2R3-MYB proteins in the regulation of distinct but interconnecting metabolism pathways.

Large-scale screening of transcription factor-promoter interactions in spruce reveals a transcriptional network involved in vascular development.

This research aimed to investigate the role of diverse transcription factors (TFs) and to delineate gene regulatory networks directly in conifers at a relatively high-throughput level. The approach integrated sequence analyses, transcript profiling, and development of a conifer-specific activation assay. Transcript accumulation profiles of 102 TFs and potential target genes were clustered to identify groups of coordinately expressed genes. Several different patterns of transcript accumulation were observed by profiling in nine different organs and tissues: 27 genes were preferential to secondary xylem both in stems and roots, and other genes were preferential to phelloderm and periderm or were more ubiquitous. A robust system has been established as a screening approach to define which TFs have the ability to regulate a given promoter in planta. Trans-activation or repression effects were observed in 30% of TF-candidate gene promoter combinations. As a proof of concept, phylogenetic analysis and expression and trans-activation data were used to demonstrate that two spruce NAC-domain proteins most likely play key roles in secondary vascular growth as observed in other plant species. This study tested many TFs from diverse families in a conifer tree species, which broadens the knowledge of promoter-TF interactions in wood development and enables comparisons of gene regulatory networks found in angiosperms and gymnosperms.

Conifers Concentrate Large Numbers of NLR Immune Receptor Genes on One Chromosome.

Nucleotide-binding domain and leucine-rich repeat (NLR) immune receptor genes form a major line of defense in plants, acting in both pathogen recognition and resistance machinery activation. NLRs are reported to form large gene clusters in limber pine (Pinus flexilis), but it is unknown how widespread this genomic architecture may be among the extant species of conifers (Pinophyta). We used comparative genomic analyses to assess patterns in the abundance, diversity, and genomic distribution of NLR genes. Chromosome-level whole genome assemblies and high-density linkage maps in the Pinaceae, Cupressaceae, Taxaceae, and other gymnosperms were scanned for NLR genes using existing and customized pipelines. The discovered genes were mapped across chromosomes and linkage groups and analyzed phylogenetically for evolutionary history. Conifer genomes are characterized by dense clusters of NLR genes, highly localized on one chromosome. These clusters are rich in TNL-encoding genes, which seem to have formed through multiple tandem duplication events. In contrast to angiosperms and nonconiferous gymnosperms, genomic clustering of NLR genes is ubiquitous in conifers. NLR-dense genomic regions are likely to influence a large part of the plant's resistance, informing our understanding of adaptation to biotic stress and the development of genetic resources through breeding.

High-density genetic linkage mapping in Sitka spruce advances the integration of genomic resources in conifers.

In species with large and complex genomes such as conifers, dense linkage maps are a useful resource for supporting genome assembly and laying the genomic groundwork at the structural, populational, and functional levels. However, most of the 600+ extant conifer species still lack extensive genotyping resources, which hampers the development of high-density linkage maps. In this study, we developed a linkage map relying on 21,570 single nucleotide polymorphism (SNP) markers in Sitka spruce (Picea sitchensis [Bong.] Carr.), a long-lived conifer from western North America that is widely planted for productive forestry in the British Isles. We used a single-step mapping approach to efficiently combine RAD-seq and genotyping array SNP data for 528 individuals from 2 full-sib families. As expected for spruce taxa, the saturated map contained 12 linkages groups with a total length of 2,142 cM. The positioning of 5,414 unique gene coding sequences allowed us to compare our map with that of other Pinaceae species, which provided evidence for high levels of synteny and gene order conservation in this family. We then developed an integrated map for P. sitchensis and Picea glauca based on 27,052 markers and 11,609 gene sequences. Altogether, these 2 linkage maps, the accompanying catalog of 286,159 SNPs and the genotyping chip developed, herein, open new perspectives for a variety of fundamental and more applied research objectives, such as for the improvement of spruce genome assemblies, or for marker-assisted sustainable management of genetic resources in Sitka spruce and related species.

Are long-lived trees poised for evolutionary change? Single locus effects in the evolution of gene expression networks in spruce.

Genetic variation in gene expression traits contributes to phenotypic diversity and may facilitate adaptation following environmental change. This is especially important in long-lived organisms where adaptation to rapid changes in the environment must rely on standing variation within populations. However, the extent of expression variation in most wild species remains to be investigated. We address this question by measuring the segregation of expression levels in white spruce [Picea glauca (Moench), Voss] in a transcriptome-wide manner and examining the underlying evolutionary and biological processes. We applied a novel approach for the genetic analysis of expression variation by measuring its segregation in haploid meiotic seed tissue. We identified over 800 transcripts whose abundances are most likely controlled by variants in single loci. Cosegregation analysis of allelic expression levels was used to construct regulatory associations between genes and define regulatory networks. The majority (67%) of segregating transcripts were under linkage. Regulatory associations were typically among small groups of genes (2-3 transcripts), indicating that most segregating expression levels can evolve independently from one another. One notable exception was a large putative trans effect that altered the expression of 180 genes that includes key regulators of protein metabolism, highlighting a regulatory cascade affected by variation in a single locus in this conserved metabolic pathway. Overall, segregating expression variation was associated with stress response- and duplicated genes, whose evolution may be linked to functional innovations. These observations indicate that expression variation might be important in facilitating diversity of molecular responses to environmental stresses in wild trees.

Comparing DNA isolation methods for forest trees: quality, plastic footprint, and time-efficiency.

BACKGROUND: Genetic and genomic studies are seeing an increase in sample sizes together with a wider range of species investigated in response to environmental change concerns. In turn, these changes may come with challenges including the time and difficulty to isolate nucleic acids (DNA or RNA), the sequencing cost and environmental impacts of the growing amount of plastic waste generated in the process. Pseudotsuga menziesii var. menziesii (Mirbel) Franco (PM), Tsuga heterophylla (Raf.) Sarg. (TH) and Thuja plicata Donn ex D.Don (TP) are conifer species found in diverse woodlands both as natives and naturalized exotics. Our study was carried out whilst investigating their genetics to understand their population structure and potential for adaptation. RESULTS: In the present study, we compared two different DNA isolation methods, i.e., spin-column DNeasy plant mini kit (QIAGEN), and temperature-driven enzymatic cocktail Plant DNA Extraction (MicroGEM). The quantity of recovered DNA and the quality of DNA were assessed along with the plastic footprint and time needed for three tree species. Both methods were optimised and proven to provide enough DNA for each studied species. The yield of DNA for each method depended on the species: QIAGEN showed higher yield in P. menziesii and T. heterophylla, while T. plicata recovered similar amount of DNA for both methods. The DNA quality was investigated using DNA barcoding techniques by confirming species identity and species discrimination. No difference was detected in the PCR amplification of the two barcoding loci, (rbcL and trnH-psbA), and the recovered sequences between DNA isolation methods. Measurement of the plastic use and the processing time per sample indicated that MicroGEM had a 52.64% lower plastic footprint and was 51.8% faster than QIAGEN. CONCLUSIONS: QIAGEN gave higher yields in two of the species although both methods showed similar quality results across all species. However, MicroGEM was clearly advantageous to decrease the plastic footprint and improve the time efficiency. Overall, MicroGEM recovers sufficient and reliable DNA to perform common downstream analyses such as PCR and sequencing. Our findings illustrate the benefits of research and efforts towards developing more sustainable methods and techniques to reduce the environmental footprint of molecular analyses.

A white spruce gene catalog for conifer genome analyses.

Several angiosperm plant genomes, including Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), poplar (Populus trichocarpa), and grapevine (Vitis vinifera), have been sequenced, but the lack of reference genomes in gymnosperm phyla reduces our understanding of plant evolution and restricts the potential impacts of genomics research. A gene catalog was developed for the conifer tree Picea glauca (white spruce) through large-scale expressed sequence tag sequencing and full-length cDNA sequencing to facilitate genome characterizations, comparative genomics, and gene mapping. The resource incorporates new and publicly available sequences into 27,720 cDNA clusters, 23,589 of which are represented by full-length insert cDNAs. Expressed sequence tags, mate-pair cDNA clone analysis, and custom sequencing were integrated through an iterative process to improve the accuracy of clustering outcomes. The entire catalog spans 30 Mb of unique transcribed sequence. We estimated that the P. glauca nuclear genome contains up to 32,520 transcribed genes owing to incomplete, partially sequenced, and unsampled transcripts and that its transcriptome could span up to 47 Mb. These estimates are in the same range as the Arabidopsis and rice transcriptomes. Next-generation methods confirmed and enhanced the catalog by providing deeper coverage for rare transcripts, by extending many incomplete clusters, and by augmenting the overall transcriptome coverage to 38 Mb of unique sequence. Genomic sample sequencing at 8.5% of the 19.8-Gb P. glauca genome identified 1,495 clusters representing highly repeated sequences among the cDNA clusters. With a conifer transcriptome in full view, functional and protein domain annotations clearly highlighted the divergences between conifers and angiosperms, likely reflecting their respective evolutionary paths.

Gene family structure, expression and functional analysis of HD-Zip III genes in angiosperm and gymnosperm forest trees.

BACKGROUND: Class III Homeodomain Leucine Zipper (HD-Zip III) proteins have been implicated in the regulation of cambium identity, as well as primary and secondary vascular differentiation and patterning in herbaceous plants. They have been proposed to regulate wood formation but relatively little evidence is available to validate such a role. We characterised and compared HD-Zip III gene family in an angiosperm tree, Populus spp. (poplar), and the gymnosperm Picea glauca (white spruce), representing two highly evolutionarily divergent groups. RESULTS: Full-length cDNA sequences were isolated from poplar and white spruce. Phylogenetic reconstruction indicated that some of the gymnosperm sequences were derived from lineages that diverged earlier than angiosperm sequences, and seem to have been lost in angiosperm lineages. Transcript accumulation profiles were assessed by RT-qPCR on tissue panels from both species and in poplar trees in response to an inhibitor of polar auxin transport. The overall transcript profiles HD-Zip III complexes in white spruce and poplar exhibited substantial differences, reflecting their evolutionary history. Furthermore, two poplar sequences homologous to HD-Zip III genes involved in xylem development in Arabidopsis and Zinnia were over-expressed in poplar plants. PtaHB1 over-expression produced noticeable effects on petiole and primary shoot fibre development, suggesting that PtaHB1 is involved in primary xylem development. We also obtained evidence indicating that expression of PtaHB1 affected the transcriptome by altering the accumulation of 48 distinct transcripts, many of which are predicted to be involved in growth and cell wall synthesis. Most of them were down-regulated, as was the case for several of the poplar HD-Zip III sequences. No visible physiological effect of over-expression was observed on PtaHB7 transgenic trees, suggesting that PtaHB1 and PtaHB7 likely have distinct roles in tree development, which is in agreement with the functions that have been assigned to close homologs in herbaceous plants. CONCLUSIONS: This study provides an overview of HD-zip III genes related to woody plant development and identifies sequences putatively involved in secondary vascular growth in angiosperms and in gymnosperms. These gene sequences are candidate regulators of wood formation and could be a source of molecular markers for tree breeding related to wood properties.

High nitrogen fertilization and stem leaning have overlapping effects on wood formation in poplar but invoke largely distinct molecular pathways.

Previous studies indicated that high nitrogen fertilization may impact secondary xylem development and alter fibre anatomy and composition. The resulting wood shares some resemblance with tension wood, which has much thicker cell walls than normal wood due to the deposition of an additional layer known as the G-layer. This report compares the short-term effects of high nitrogen fertilization and tree leaning to induce tension wood, either alone or in combination, upon wood formation in young trees of Populus trichocarpa (Torr. & Gray) × P. deltoides Bartr. ex Marsh. Fibre anatomy, chemical composition and transcript profiles were examined in newly formed secondary xylem. Each of the treatments resulted in thicker cell walls relative to the controls. High nitrogen and tree leaning had overlapping effects on chemical composition based on Fourier transform infrared analysis, specifically indicating that secondary cell wall composition was shifted in favour of cellulose and hemicelluloses relative to lignin content. In contrast, the high-nitrogen trees had shorter fibres, whilst the leaning trees had longer fibres that the controls. Microarray transcript profiling carried out after 28 days of treatment identified 180 transcripts that accumulated differentially in one or more treatments. Only 10% of differentially expressed transcripts were affected in all treatments relative to the controls. Several of the affected transcripts were related to carbohydrate metabolism, secondary cell wall formation, nitrogen metabolism and osmotic stress. RT-qPCR analyses at 1, 7 and 28 days showed that several transcripts followed very different accumulation profiles in terms of rate and level of accumulation, depending on the treatment. Our findings suggest that high nitrogen fertilization and tension wood induction elicit largely distinct and molecular pathways with partial overlap. When combined, the two types of environmental cue yielded additive effects.

Reference transcriptomes and comparative analyses of six species in the threatened rosewood genus Dalbergia.

Dalbergia is a pantropical genus with more than 250 species, many of which are highly threatened due to overexploitation for their rosewood timber, along with general deforestation. Many Dalbergia species have received international attention for conservation, but the lack of genomic resources for Dalbergia hinders evolutionary studies and conservation applications, which are important for adaptive management. This study produced the first reference transcriptomes for 6 Dalbergia species with different geographical origins and predicted ~ 32 to 49 K unique genes. We showed the utility of these transcriptomes by phylogenomic analyses with other Fabaceae species, estimating the divergence time of extant Dalbergia species to ~ 14.78 MYA. We detected over-representation in 13 Pfam terms including HSP, ALDH and ubiquitin families in Dalbergia. We also compared the gene families of geographically co-occurring D. cochinchinensis and D. oliveri and observed that more genes underwent positive selection and there were more diverged disease resistance proteins in the more widely distributed D. oliveri, consistent with reports that it occupies a wider ecological niche and has higher genetic diversity. We anticipate that the reference transcriptomes will facilitate future population genomics and gene-environment association studies on Dalbergia, as well as contributing to the genomic database where plants, particularly threatened ones, are currently underrepresented.