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Our study utilized genome-wide association studies (GWAS) to link nucleotide variants to traits in Populus trichocarpa, a species with rapid linkage disequilibrium decay. The aim was to overcome the challenge of interpreting statistical associations at individual loci without sufficient biological context, which often leads to reliance solely on gene annotations from unrelated model organisms. We employed an integrative approach that included GWAS targeting multiple traits using three individual techniques for lignocellulose phenotyping, expression quantitative trait loci (eQTL) analysis to construct transcriptional regulatory networks around each candidate locus and co-expression analysis to provide biological context for these networks, using lignocellulose biosynthesis in Populus trichocarpa as a case study. The research identified three candidate genes potentially involved in lignocellulose formation, including one previously recognized gene (Potri.005G116800/VND1, a critical regulator of secondary cell wall formation) and two genes (Potri.012G130000/AtSAP9 and Potri.004G202900/BIC1) with newly identified putative roles in lignocellulose biosynthesis. Our integrative approach offers a framework for providing biological context to loci associated with trait variation, facilitating the discovery of new genes and regulatory networks., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Community genetics seeks to understand the mechanisms by which natural genetic variation in heritable host phenotypes can encompass assemblages of organisms such as bacteria, fungi, and many animals including arthropods. Prior studies that focused on plant genotypes have been unable to identify genes controlling community composition, a necessary step to predict ecosystem structure and function as underlying genes shift within plant populations. We surveyed arthropods within an association population of Populus trichocarpa in three common gardens to discover plant genes that contributed to arthropod community composition. We analyzed our surveys with traditional single-trait genome-wide association analysis (GWAS), multitrait GWAS, and functional networks built from a diverse set of plant phenotypes. Plant genotype was influential in structuring arthropod community composition among several garden sites. Candidate genes important for higher level organization of arthropod communities had broadly applicable functions, such as terpenoid biosynthesis and production of dsRNA binding proteins and protein kinases, which may be capable of targeting multiple arthropod species. We have demonstrated the ability to detect, in an uncontrolled environment, individual genes that are associated with the community assemblage of arthropods on a host plant, further enhancing our understanding of genetic mechanisms that impact ecosystem structure., (© 2024 UT‐Battelle, LLC, and The Authors. New Phytologist © 2024 New Phytologist Foundation.)

Plant regeneration is an important dimension of plant propagation and a key step in the production of transgenic plants. However, regeneration capacity varies widely among genotypes and species, the molecular basis of which is largely unknown. Association mapping methods such as genome-wide association studies (GWAS) have long demonstrated abilities to help uncover the genetic basis of trait variation in plants; however, the performance of these methods depends on the accuracy and scale of phenotyping. To enable a large-scale GWAS of in planta callus and shoot regeneration in the model tree Populus, we developed a phenomics workflow involving semantic segmentation to quantify regenerating plant tissues over time. We found that the resulting statistics were of highly non-normal distributions, and thus employed transformations or permutations to avoid violating assumptions of linear models used in GWAS. We report over 200 statistically supported quantitative trait loci (QTLs), with genes encompassing or near to top QTLs including regulators of cell adhesion, stress signaling, and hormone signaling pathways, as well as other diverse functions. Our results encourage models of hormonal signaling during plant regeneration to consider keystone roles of stress-related signaling (e.g. involving jasmonates and salicylic acid), in addition to the auxin and cytokinin pathways commonly considered. The putative regulatory genes and biological processes we identified provide new insights into the biological complexity of plant regeneration, and may serve as new reagents for improving regeneration and transformation of recalcitrant genotypes and species., Competing Interests: Conflicts of interest. The author(s) declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Plant lignocellulosic biomass, i.e. secondary cell walls of plants, is a vital alternative source for bioenergy. However, the acetylation of xylan in secondary cell walls impedes the conversion of biomass to biofuels. Previous studies have shown that REDUCED WALL ACETYLATION (RWA) proteins are directly involved in the acetylation of xylan but the regulatory mechanism of RWAs is not fully understood. In this study, we demonstrate that overexpression of a Populus trichocarpa PtRWA-C gene increases the level of xylan acetylation and increases the lignin content and S/G ratio, ultimately yielding poplar woody biomass with reduced saccharification efficiency. Furthermore, through gene coexpression network and expression quantitative trait loci (eQTL) analysis, we found that PtRWA-C was regulated not only by the secondary cell wall hierarchical regulatory network but also by an AP2 family transcription factor HARDY (HRD). Specifically, HRD activates PtRWA-C expression by directly binding to the PtRWA-C promoter, which is also the cis-eQTL for PtRWA-C. Taken together, our findings provide insights into the functional roles of PtRWA-C in xylan acetylation and consequently saccharification and shed light on synthetic biology approaches to manipulate this gene and alter cell wall properties. These findings have substantial implications for genetic engineering of woody species, which could be used as a sustainable source of biofuels, valuable biochemicals, and biomaterials., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Plant establishment requires the formation and development of an extensive root system with architecture modulated by complex genetic networks. Here, we report the identification of the PtrXB38 gene as an expression quantitative trait loci (eQTL) hotspot, mapped using 390 leaf and 444 xylem Populus trichocarpa transcriptomes. Among predicted targets of this trans-eQTL were genes involved in plant hormone responses and root development. Overexpression of PtrXB38 in Populus led to significant increases in callusing and formation of both stem-born roots and base-born adventitious roots. Omics studies revealed that genes and proteins controlling auxin transport and signaling were involved in PtrXB38-mediated adventitious root formation. Protein-protein interaction assays indicated that PtrXB38 interacts with components of endosomal sorting complexes required for transport machinery, implying that PtrXB38-regulated root development may be mediated by regulating endocytosis pathway. Taken together, this work identified a crucial root development regulator and sheds light on the discovery of other plant developmental regulators through combining eQTL mapping and omics approaches., (© 2023 The Authors New Phytologist © 2023 New Phytologist Foundation.)

The placenta in fruits of most plants either desiccate and shrink as the fruits mature or develop further to form the fleshy tissues. In poplars, placental epidermal cells protrude collectively to produce catkin fibers. In this study, three carpel limited MIXTA genes, PdeMIXTA02, PdeMIXTA03, PdeMIXTA04, were find to specifically expressed in carpel immediately after pollination. Heterologous expression of the three genes in Arabidopsis demonstrated that PdeMIXTA04 significantly promoted trichomes density and could restore trichomes in the trichomeless mutant. By contrast, such functions were not observed with PdeMIXTA02, PdeMIXTA03. In situ hybridization revealed that PdeMIXTA04 was explicitly expressed in poplar placental epidermal cells. We also confirmed trichome-specific expression of the PdeMIXTA04 promoter. Multiple experimental proofs have confirmed the interaction between PdeMIXTA04, PdeMYC and PdeWD40, indicating PdeMIXTA04 functioned through the MYB-bHLH-WD40 ternary complex. Our work provided distinctive understanding of the molecular mechanism triggering differentiation of poplar catkins., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Deciduous woody plants like poplar (Populus spp.) have seasonal bud dormancy. It has been challenging to simultaneously delay the onset of bud dormancy in the fall and advance bud break in the spring, as bud dormancy, and bud break were thought to be controlled by different genetic factors. Here, we demonstrate that heterologous expression of the REVEILLE1 gene (named AaRVE1) from Agave (Agave americana) not only delays the onset of bud dormancy but also accelerates bud break in poplar in field trials. AaRVE1 heterologous expression increases poplar biomass yield by 166% in the greenhouse. Furthermore, we reveal that heterologous expression of AaRVE1 increases cytokinin contents, represses multiple dormancy-related genes, and up-regulates bud break-related genes, and that AaRVE1 functions as a transcriptional repressor and regulates the activity of the DORMANCY-ASSOCIATED PROTEIN 1 (DRM1) promoter. Our findings demonstrate that AaRVE1 appears to function as a regulator of bud dormancy and bud break, which has important implications for extending the growing season of deciduous trees in frost-free temperate and subtropical regions to increase crop yield., Competing Interests: Conflict of interest statement. Authors declare no conflict of interest., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Fine-scale meiotic recombination is fundamental to the outcome of natural and artificial selection. Here, dense genetic mapping and haplotype reconstruction were used to estimate recombination for a full factorial Populus trichocarpa cross of 7 males and 7 females. Genomes of the resulting 49 full-sib families (N = 829 offspring) were resequenced, and high-fidelity biallelic SNP/INDELs and pedigree information were used to ascertain allelic phase and impute progeny genotypes to recover gametic haplotypes. The 14 parental genetic maps contained 1,820 SNP/INDELs on average that covered 376.7 Mb of physical length across 19 chromosomes. Comparison of parental and progeny haplotypes allowed fine-scale demarcation of cross-over regions, where 38,846 cross-over events in 1,658 gametes were observed. Cross-over events were positively associated with gene density and negatively associated with GC content and long-terminal repeats. One of the most striking findings was higher rates of cross-overs in males in 8 out of 19 chromosomes. Regions with elevated male cross-over rates had lower gene density and GC content than windows showing no sex bias. High-resolution analysis identified 67 candidate cross-over hotspots spread throughout the genome. DNA sequence motifs enriched in these regions showed striking similarity to those of maize, Arabidopsis, and wheat. These findings, and recombination estimates, will be useful for ongoing efforts to accelerate domestication of this and other biomass feedstocks, as well as future studies investigating broader questions related to evolutionary history, perennial development, phenology, wood formation, vegetative propagation, and dioecy that cannot be studied using annual plant model systems., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.)

Leaf morphogenesis requires precise regulation of gene expression to achieve organ separation and flat-leaf form. The poplar KNOTTED-like homeobox gene PagKNAT2/6b could change plant architecture, especially leaf shape, in response to drought stress. However, its regulatory mechanism in leaf development remains unclear. In this work, gene expression analyses of PagKNAT2/6b suggested that PagKNAT2/6b was highly expressed during leaf development. Moreover, the leaf shape changes along the adaxial-abaxial, medial-lateral, and proximal-distal axes caused by the mis-expression of PagKNAT2/6b demonstrated that its overexpression ( PagKNAT2/6b OE) and SRDX dominant repression ( PagKNAT2/6b SRDX) poplars had an impact on the leaf axial development. The crinkle leaf of PagKNAT2/6b OE was consistent with the differential expression gene PagBOP1/2a ( BLADE-ON-PETIOLE ), which was the critical gene for regulating leaf development. Further study showed that PagBOP1/2a was directly activated by PagKNAT2/6b through a novel cis -acting element "CTCTT". Together, the PagKNAT2/6b-PagBOP1/2a module regulates poplar leaf morphology by affecting axial development, which provides insights aimed at leaf shape modification for further improving the drought tolerance of woody plants.

Populus spp. are dedicated woody biomass feedstocks for advanced biofuels and bioproducts. Proper growth and fitness of poplar as a sustainable feedstock depends on timely perception and response to environmental signals (e.g., light, temperature, water). Poplar leaves, like other C3 photosynthesis plants, have evolved oscillating or circadian rhythms that play important roles in synchronizing biological processes with external cues. To characterize this phenomenon at a molecular level, we employed bottom-up proteomics using high-resolution mass spectrometry and de novo-assisted database searching to identify abundance changes in proteins and post-translational modifications in poplar leaf tissue sampled across a 12/12-hour light/dark diurnal period., (© 2021 Wiley-VCH GmbH.)

Orphan genes are characteristic genomic features that have no detectable homology to genes in any other species and represent an important attribute of genome evolution as sources of novel genetic functions. Here, we identified 445 genes specific to Populus trichocarpa. Of these, we performed deeper reconstruction of 13 orphan genes to provide evidence of de novo gene evolution. Populus and its sister genera Salix are particularly well suited for the study of orphan gene evolution because of the Salicoid whole-genome duplication event which resulted in highly syntenic sister chromosomal segments across the Salicaceae. We leveraged this genomic feature to reconstruct de novo gene evolution from intergenera, interspecies, and intragenomic perspectives by comparing the syntenic regions within the P. trichocarpa reference, then P. deltoides, and finally Salix purpurea. Furthermore, we demonstrated that 86.5% of the putative orphan genes had evidence of transcription. Additionally, we also utilized the Populus genome-wide association mapping panel, a collection of 1,084 undomesticated P. trichocarpa genotypes to further determine putative regulatory networks of orphan genes using expression quantitative trait loci (eQTL) mapping. Functional enrichment of these eQTL subnetworks identified common biological themes associated with orphan genes such as response to stress and defense response. We also identify a putative cis-element for a de novo gene and leverage conserved synteny to describe evolution of a putative transcription factor binding site. Overall, 45% of orphan genes were captured in trans-eQTL networks., (Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution 2021. This work is written by a US Government employee and is in the public domain in the US.)

Background: Plants can transmit somatic mutations and epimutations to offspring, which in turn can affect fitness. Knowledge of the rate at which these variations arise is necessary to understand how plant development contributes to local adaption in an ecoevolutionary context, particularly in long-lived perennials., Results: Here, we generate a new high-quality reference genome from the oldest branch of a wild Populus trichocarpa tree with two dominant stems which have been evolving independently for 330 years. By sampling multiple, age-estimated branches of this tree, we use a multi-omics approach to quantify age-related somatic changes at the genetic, epigenetic, and transcriptional level. We show that the per-year somatic mutation and epimutation rates are lower than in annuals and that transcriptional variation is mainly independent of age divergence and cytosine methylation. Furthermore, a detailed analysis of the somatic epimutation spectrum indicates that transgenerationally heritable epimutations originate mainly from DNA methylation maintenance errors during mitotic rather than during meiotic cell divisions., Conclusion: Taken together, our study provides unprecedented insights into the origin of nucleotide and functional variation in a long-lived perennial plant.

Members of the genus Populus (i.e., cottonwood, hybrid poplar) represent a promising source of lignocellulosic biomass for biofuels. However, one of the major factors negatively affecting poplar's efficient conversion to biofuel is the inherent recalcitrance to enzymatic saccharification due to cell wall components such as lignin. To this effect, there have been efforts to modify gene expression to reduce biomass recalcitrance by changing cell wall properties. Here, we review recent genetic modifications of poplar that led to change cell wall properties and the resulting effects on subsequent pretreatment efficacy and saccharification. Although genetic engineering's impacts on cell wall properties are not fully predictable, recent studies have shown promising improvement in the biological conversion of transgenic poplar to biofuels., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The ages and sizes of a sex-determination region (SDR) are difficult to determine in non-model species. Due to the lack of recombination and enrichment of repetitive elements in SDRs, the quality of assembly with short sequencing reads is universally low. Unique features present in the SDRs help provide clues about how SDRs are established and how they evolve in the absence of recombination. Several Populus species have been reported with a male heterogametic configuration of sex (XX/XY system) mapped on chromosome 19, but the exact location of the SDR has been inconsistent among species, and thus far, none of these SDRs has been fully assembled in a genomic context. Here we identify the Y-SDR from a Y-linked contig directly from a long-read PacBio assembly of a Populus trichocarpa male individual. We also identified homologous gene sequences in the SDR of P. trichocarpa and the SDR of the W chromosome in Salix purpurea . We show that inverted repeats (IRs) found in the Y-SDR and the W-SDR are lineage-specific. We hypothesize that, although the two IRs are derived from the same orthologous gene within each species, they likely have independent evolutionary histories. Furthermore, the truncated inverted repeats in P. trichocarpa may code for small RNAs that target the homologous gene for RNA-directed DNA methylation. These findings support the hypothesis that diverse sex-determining systems may be achieved through similar evolutionary pathways, thereby providing a possible mechanism to explain the lability of sex-determination systems in plants in general.

Prefoldin (PFD) is a group II chaperonin that is ubiquitously present in the eukaryotic kingdom. Six subunits (PFD1-6) form a jellyfish-like heterohexameric PFD complex and function in protein folding and cytoskeleton organization. However, little is known about its function in plant cell wall-related processes. Here, we report the functional characterization of a PFD gene from Populus deltoides, designated as PdPFD2.2. There are two copies of PFD2 in Populus, and PdPFD2.2 was ubiquitously expressed with high transcript abundance in the cambial region. PdPFD2.2 can physically interact with DELLA protein RGA1_8g, and its subcellular localization is affected by the interaction. In P. deltoides transgenic plants overexpressing PdPFD2.2, the lignin syringyl/guaiacyl ratio was increased, but cellulose content and crystallinity index were unchanged. In addition, the total released sugar (glucose and xylose) amounts were increased by 7.6% and 6.1%, respectively, in two transgenic lines. Transcriptomic and metabolomic analyses revealed that secondary metabolic pathways, including lignin and flavonoid biosynthesis, were affected by overexpressing PdPFD2.2. A total of eight hub transcription factors (TFs) were identified based on TF binding sites of differentially expressed genes in Populus transgenic plants overexpressing PdPFD2.2. In addition, several known cell wall-related TFs, such as MYB3, MYB4, MYB7, TT8 and XND1, were affected by overexpression of PdPFD2.2. These results suggest that overexpression of PdPFD2.2 can reduce biomass recalcitrance and PdPFD2.2 is a promising target for genetic engineering to improve feedstock characteristics to enhance biofuel conversion and reduce the cost of lignocellulosic biofuel production., (© 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

In the present study, three leading pretreatment technologies including dilute acid (DA), liquid hot water (LHW), and organosolv pretreatments (OS) were applied on two Populus natural variants with different recalcitrance. The structural features of the isolated lignins were analyzed accordingly. All the studied pretreatments reduced the molecular weights of the lignins. Aliphatic OH was reduced while phenolic OH was increased in all pretreated lignins. HSQC analysis revealed that pretreatment influenced the lignin composition and relative distribution of inter-unit linkages. The lignin S/G ratio was found to increase during DA pretreatment, while it was decreased after LHW and OS pretreatment. LHW pretreatment also resulted in much less cleavage of β-O-4 linkage than the other two pretreatments. These results could offer guidelines on appropriate selection of biomass and pretreatment technology in the future biorefinery process., (Copyright © 2019. Published by Elsevier Ltd.)

Background: Plant secondary cell wall is a renewable feedstock for biofuels and biomaterials production. Arabidopsis VASCULAR-RELATED NAC DOMAIN (VND) has been demonstrated to be a key transcription factor regulating secondary cell wall biosynthesis. However, less is known about its role in the woody species., Results: Here we report the functional characterization of Populus deltoides WOOD-ASSOCIATED NAC DOMAIN protein 3 (PdWND3A), a sequence homolog of Arabidopsis VND4 and VND5 that are members of transcription factor networks regulating secondary cell wall biosynthesis. PdWND3A was expressed at higher level in the xylem than in other tissues. The stem tissues of transgenic P. deltoides overexpressing PdWND3A (OXPdWND3A) contained more vessel cells than that of wild-type plants. Furthermore, lignin content and lignin monomer syringyl and guaiacyl (S/G) ratio were higher in OXPdWND3A transgenic plants than in wild-type plants. Consistent with these observations, the expression of FERULATE 5-HYDROXYLASE1 (F5H1), encoding an enzyme involved in the biosynthesis of sinapyl alcohol (S unit monolignol), was elevated in OXPdWND3A transgenic plants. Saccharification analysis indicated that the rate of sugar release was reduced in the transgenic plants. In addition, OXPdWND3A transgenic plants produced lower amounts of biomass than wild-type plants., Conclusions: PdWND3A affects lignin biosynthesis and composition and negatively impacts sugar release and biomass production.

Background and Aims: The use of woody crops for Quad-level (approx. 1 × 1018 J) energy production will require marginal agricultural lands that experience recurrent periods of water stress. Populus species have the capacity to increase dehydration tolerance by lowering osmotic potential via osmotic adjustment. The aim of this study was to investigate how the inherent genetic potential of a Populus clone to respond to drought interacts with the nature of the drought to determine the degree of biochemical response., Methods: A greenhouse drought stress study was conducted on Populus deltoides 'WV94' and the resulting metabolite profiles of leaves were determined by gas chromatography-mass spectrometry following trimethylsilylation for plants subjected to cyclic mild (-0.5 MPa pre-dawn leaf water potential) drought vs. cyclic severe (-1.26 MPa) drought in contrast to well-watered controls (-0.1 MPa) after two or four drought cycles, and in contrast to plants subjected to acute drought, where plants were desiccated for up to 8 d., Key Results: The nature of drought (cyclic vs. acute), frequency of drought (number of cycles) and the severity of drought (mild vs. severe) all dictated the degree of osmotic adjustment and the nature of the organic solutes that accumulated. Whereas cyclic drought induced the largest responses in primary metabolism (soluble sugars, organic acids and amino acids), acute onset of prolonged drought induced the greatest osmotic adjustment and largest responses in secondary metabolism, especially populosides (hydroxycinnamic acid conjugates of salicin)., Conclusions: The differential adaptive metabolite responses in cyclic vs. acute drought suggest that stress acclimation occurs via primary metabolism in response to cyclic drought, whereas expanded metabolic plasticity occurs via secondary metabolism following severe, acute drought. The shift in carbon partitioning to aromatic metabolism with the production of a diverse suite of higher order salicylates lowers osmotic potential and increases the probability of post-stress recovery., (Published by Oxford University Press on behalf of the Annals of Botany Company 2019.)

Pyrolysis of five poplar samples with differing degrees of recalcitrance was performed; the correlations between the poplar enzymatic hydrolysis glucose yields and the physicochemical properties of pyrolysis product were investigated in this study. Sugar release of five poplar samples varied from 48.1 to 112.3 mg/g for glucose, and 12.0 to 32.4 mg/g for xylose. The yield of pyrolysis products was calculated and the molecular weight distribution of pyrolysis oils was measured by GPC, ranging from 268 to 289 g/mol for its weight-average molecular weight. GC-MS analysis of the bio-oil exhibited a strong correlation between biomass recalcitrance and guaiacyl-type structures in bio-oils. The correlation between biomass recalcitrance and the ratio of syringyl-to-guaiacyl-type-related structures was also assessed. The results from quantitative 31 P NMR indicated some correlation between biomass recalcitrance and the guaiacyl hydroxyl groups in bio-oils. These results illustrate correlations and differences between converting biomass to biofuels via the biological and thermal platform., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Mortierella and Ilyonectria genera include common species of soil fungi that are frequently detected as root endophytes in many plants, including Populus spp. However, the ecological roles of these and other endophytic fungi with respect to plant growth and function are still not well understood. The functional ecology of two key taxa from the P. trichocarpa rhizobiome, M. elongata PMI93 and I. europaea PMI82, was studied by coupling forest soil bioassays with environmental metatranscriptomics. Using soil bioassay experiments amended with fungal inoculants, M. elongata was observed to promote the growth of P. trichocarpa . This response was cultivar independent. In contrast, I. europaea had no visible effect on P. trichocarpa growth. Metatranscriptomic studies revealed that these fungi impacted rhizophytic and endophytic activities in P. trichocarpa and induced shifts in soil and root microbial communities. Differential expression of core genes in P. trichocarpa roots was observed in response to both fungal species. Expression of P. trichocarpa genes for lipid signaling and nutrient uptake were upregulated, and expression of genes associated with gibberellin signaling were altered in plants inoculated with M. elongata , but not I. europaea . Upregulation of genes for growth promotion, downregulation of genes for several leucine-rich repeat receptor kinases, and alteration of expression of genes associated with plant defense responses (e.g., jasmonic acid, salicylic acid, and ethylene signal pathways) also suggest that M. elongata manipulates plant defenses while promoting plant growth.

Genome-wide association studies (GWAS) have great promise for identifying the loci that contribute to adaptive variation, but the complex genetic architecture of many quantitative traits presents a substantial challenge. We measured 14 morphological and physiological traits and identified single nucleotide polymorphism (SNP)-phenotype associations in a Populus trichocarpa population distributed from California, USA to British Columbia, Canada. We used whole-genome resequencing data of 882 trees with more than 6.78 million SNPs, coupled with multitrait association to detect polymorphisms with potentially pleiotropic effects. Candidate genes were validated with functional data. Broad-sense heritability (H 2 ) ranged from 0.30 to 0.56 for morphological traits and 0.08 to 0.36 for physiological traits. In total, 4 and 20 gene models were detected using the single-trait and multitrait association methods, respectively. Several of these associations were corroborated by additional lines of evidence, including co-expression networks, metabolite analyses, and direct confirmation of gene function through RNAi. Multitrait association identified many more significant associations than single-trait association, potentially revealing pleiotropic effects of individual genes. This approach can be particularly useful for challenging physiological traits such as water-use efficiency or complex traits such as leaf morphology, for which we were able to identify credible candidate genes by combining multitrait association with gene co-expression and co-methylation data., (No claim to US Government works New Phytologist © 2019 New Phytologist Trust.)

The molecular mechanisms underlying mycorrhizal symbioses, the most ubiquitous and impactful mutualistic plant-microbial interaction in nature, are largely unknown. Through genetic mapping, resequencing and molecular validation, we demonstrate that a G-type lectin receptor-like kinase (lecRLK) mediates the symbiotic interaction between Populus and the ectomycorrhizal fungus Laccaria bicolor. This finding uncovers an important molecular step in the establishment of symbiotic plant-fungal associations and provides a molecular target for engineering beneficial mycorrhizal relationships.

Invasive microbes causing diseases such as sudden oak death negatively affect ecosystems and economies around the world. The deployment of resistant genotypes for combating introduced diseases typically relies on breeding programs that can take decades to complete. To demonstrate how this process can be accelerated, we employed a genome-wide association mapping of ca 1,000 resequenced Populus trichocarpa trees individually challenged with Sphaerulina musiva , an invasive fungal pathogen. Among significant associations, three loci associated with resistance were identified and predicted to encode one putative membrane-bound L-type receptor-like kinase and two receptor-like proteins. A susceptibility-associated locus was predicted to encode a putative G-type D-mannose-binding receptor-like kinase. Multiple lines of evidence, including allele analysis, transcriptomics, binding assays, and overexpression, support the hypothesized function of these candidate genes in the P. trichocarpa response to S. musiva ., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Key Message: WUSCHEL-RELATED HOMEOBOX 11 establishes the acquisition of pluripotency during callus formation and accomplishes de novo shoot formation by regulating key transcription factors in poplar. De novo shoot regeneration is a prerequisite for propagation and genetic engineering of elite cultivars in forestry. However, the regulatory mechanism of de novo organogenesis is poorly understood in tree species. We previously showed that WUSCHEL (WUS)-RELATED HOMEOBOX 11 (PtWOX11) of the hybrid poplar clone 84K (Populus alba × P. glandulosa) promotes de novo root formation. In this study, we found that PtWOX11 also regulates de novo shoot regeneration in poplar. The overexpression of PtWOX11 enhanced de novo shoot formation, whereas overexpression of PtWOX11 fused with the transcriptional repressor domain (PtWOX11-SRDX) or reduced expression of PtWOX11 inhibited this process, indicating that PtWOX11 promotes de novo shoot organogenesis. Although PtWOX11 promotes callus formation, overexpression of PtWOX11 and PtWOX11-SRDX also produced increased and decreased numbers of de novo shoots per unit weight, respectively, implying that PtWOX11 promotes de novo shoot organogenesis partially by regulating the intrinsic mechanism of shoot development. RNA-seq and qPCR analysis further revealed that PtWOX11 activates the expression of PLETHORA1 (PtPLT1) and PtPLT2, whose Arabidopsis paralogs establish the acquisition of pluripotency, during incubation on callus-inducing medium. Moreover, PtWOX11 activates the expression of shoot-promoting factors and meristem regulators such as CUP-SHAPED COTYLEDON2 (PtCUC2), PtCUC3, WUS and SHOOT MERISTEMLESS to fulfill shoot regeneration during incubation on shoot-inducing medium. These results suggest that PtWOX11 acts as a central regulator of the expression of key genes to cause de novo shoot formation. Our studies further provide a possible means to genetically engineer economically important tree species for their micropropagation.

Four different pretreatment technologies were applied to two Populus natural variants and the effects of each pretreatment on glucose release were compared. Physicochemical properties of pretreated biomass were analyzed by attenuated total reflection Fourier transform infrared spectroscopy, gel permeation chromatography, and cross polarization/magic angle spinning carbon-13 nuclear magnetic resonance techniques. The results revealed that hemicellulose and lignin were removed to different extents during various pretreatments. The degree of polymerization of cellulose was decreased in the order of alkali > hydrothermal > organosolv > dilute acid pretreatment. Cellulose crystallinity index was slightly increased after each pretreatment. The results also demonstrated that organosolv pretreatment resulted in the highest glucose yield. Among the tested properties of Populus, degree of polymerization of cellulose was negatively correlated with glucose release, whereas hemicellulose and lignin removal, and cellulose accessibility were positively associated with glucose release from the two Populus natural variants., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

3-O-caffeoylquinic acid, also known as chlorogenic acid (CGA), functions as an intermediate in lignin biosynthesis in the phenylpropanoid pathway. It is widely distributed among numerous plant species and acts as an antioxidant in both plants and animals. Using GC-MS, we discovered consistent and extreme variation in CGA content across a population of 739 4-yr-old Populus trichocarpa accessions. We performed genome-wide association studies (GWAS) from 917 P. trichocarpa accessions and expression-based quantitative trait loci (eQTL) analyses to identify key regulators. The GWAS and eQTL analyses resolved an overlapped interval encompassing a hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase 2 (PtHCT2) that was significantly associated with CGA and partially characterized metabolite abundances. PtHCT2 leaf expression was significantly correlated with CGA abundance and it was regulated by cis-eQTLs containing W-box for WRKY binding. Among all nine PtHCT homologs, PtHCT2 is the only one that responds to infection by the fungal pathogen Sphaerulina musiva (a Populus pathogen). Validation using protoplast-based transient expression system suggests that PtHCT2 is regulated by the defense-responsive WRKY. These results are consistent with reports of CGA functioning as an antioxidant in response to biotic stress. This study provides insights into data-driven and omics-based inference of gene function in woody species., (No claim to original US government works New Phytologist © 2018 New Phytologist Trust.)

A characteristic feature of plant cells is the ability to form callus from parenchyma cells in response to biotic and abiotic stimuli. Tissue culture propagation of recalcitrant plant species and genetic engineering for desired phenotypes typically depends on efficient in vitro callus generation. Callus formation is under genetic regulation, and consequently, a molecular understanding of this process underlies successful generation for propagation materials and/or introduction of genetic elements in experimental or industrial applications. Herein, we identified 11 genetic loci significantly associated with callus formation in Populus trichocarpa using a genome-wide association study (GWAS) approach. Eight of the 11 significant gene associations were consistent across biological replications, exceeding a chromosome-wide-log10 (p) = 4.46 [p = 3.47E-05] Bonferroni-adjusted significance threshold. These eight genes were used as hub genes in a high-resolution co-expression network analysis to gain insight into the genome-wide basis of callus formation. A network of positively and negatively co-expressed genes, including several transcription factors, was identified. As proof-of-principle, a transient protoplast assay confirmed the negative regulation of a Chloroplast Nucleoid DNA-binding-related gene (Potri.018G014800) by the LEC2 transcription factor. Many of the candidate genes and co-expressed genes were 1) linked to cell division and cell cycling in plants and 2) showed homology to tumor and cancer-related genes in humans. The GWAS approach based on a high-resolution marker set, and the ability to manipulate targets genes in vitro, provided a catalog of high-confidence genes linked to callus formation that can serve as an important resource for successful manipulation of model and non-model plant species, and likewise, suggests a robust method of discovering common homologous functions across organisms., Competing Interests: The authors have declared that no competing interests exist.

Polyglutamine (polyQ) stretches have been reported to occur in proteins across many organisms including animals, fungi and plants. Expansion of these repeats has attracted much attention due their associations with numerous human diseases including Huntington's and other neurological maladies. This suggests that the relative length of polyQ stretches is an important modulator of their function. Here, we report the identification of a Populus C-terminus binding protein (CtBP) ANGUSTIFOLIA ( PtAN1 ) which contains a polyQ stretch whose functional relevance had not been established. Analysis of 917 resequenced Populus trichocarpa genotypes revealed three allelic variants at this locus encoding 11-, 13- and 15-glutamine residues. Transient expression assays using Populus leaf mesophyll protoplasts revealed that the 11Q variant exhibited strong nuclear localization whereas the 15Q variant was only found in the cytosol, with the 13Q variant exhibiting localization in both subcellular compartments. We assessed functional implications by evaluating expression changes of putative PtAN1 targets in response to overexpression of the three allelic variants and observed allele-specific differences in expression levels of putative targets. Our results provide evidence that variation in polyQ length modulates Pt AN1 function by altering subcellular localization., (Copyright © 2018 Bryan et al.)

Long-lived perennial plants, with distinctive habits of inter-annual growth, defense, and physiology, are of great economic and ecological importance. However, some biological mechanisms resulting from genome duplication and functional divergence of genes in these systems remain poorly studied. Here, we discovered an association between a poplar ( Populus trichocarpa) 5-enolpyruvylshikimate 3-phosphate synthase gene ( PtrEPSP ) and lignin biosynthesis. Functional characterization of PtrEPSP revealed that this isoform possesses a helix-turn-helix motif in the N terminus and can function as a transcriptional repressor that regulates expression of genes in the phenylpropanoid pathway in addition to performing its canonical biosynthesis function in the shikimate pathway. We demonstrated that this isoform can localize in the nucleus and specifically binds to the promoter and represses the expression of a SLEEPER -like transcriptional regulator, which itself specifically binds to the promoter and represses the expression of PtrMYB021 (known as MYB46 in Arabidopsis thaliana ), a master regulator of the phenylpropanoid pathway and lignin biosynthesis. Analyses of overexpression and RNAi lines targeting PtrEPSP confirmed the predicted changes in PtrMYB021 expression patterns. These results demonstrate that PtrEPSP in its regulatory form and PtrhAT form a transcriptional hierarchy regulating phenylpropanoid pathway and lignin biosynthesis in Populus ., (© 2018 American Society of Plant Biologists. All rights reserved.)

Drought stress is a recurring feature of world climate and the single most important factor influencing agricultural yield worldwide. Plants display highly variable, species-specific responses to drought and these responses are multifaceted, requiring physiological and morphological changes influenced by genetic and molecular mechanisms. Moreover, the reproducibility of water deficit studies is very cumbersome, which significantly impedes research on drought tolerance, because how a plant responds is highly influenced by the timing, duration, and intensity of the water deficit. Despite progress in the identification of drought-related mechanisms in many plants, the molecular basis of drought resistance remains to be fully understood in trees, particularly in poplar species because their wide geographic distribution results in varying tolerances to drought. Herein, we aimed to better understand this complex phenomenon in eastern cottonwood (Populus deltoides) by performing a detailed contrast of the proteome changes between two different water deficit experiments to identify functional intersections and divergences in proteome responses. We investigated plants subjected to cyclic water deficit and compared these responses to plants subjected to prolonged acute water deficit. In total, we identified 108,012 peptide sequences across both experiments that provided insight into the quantitative state of 22,737 Populus gene models and 8,199 functional protein groups in response to drought. Together, these datasets provide the most comprehensive insight into proteome drought responses in poplar to date and a direct proteome comparison between short period dehydration shock and cyclic, post-drought re-watering. Overall, this investigation provides novel insights into drought avoidance mechanisms that are distinct from progressive drought stress. Additionally, we identified proteins that have been associated as drought-relevant in previous studies. Importantly, we highlight the RD26 transcription factor as a gene regulated at both the transcript and protein level, regardless of species and drought condition, and, thus, represents a key, universal drought marker for Populus species.

Background: Microorganisms serve important functions within numerous eukaryotic host organisms. An understanding of the variation in the plant niche-level microbiome, from rhizosphere soils to plant canopies, is imperative to gain a better understanding of how both the structural and functional processes of microbiomes impact the health of the overall plant holobiome. Using Populus trees as a model ecosystem, we characterized the archaeal/bacterial and fungal microbiome across 30 different tissue-level niches within replicated Populus deltoides and hybrid Populus trichocarpa × deltoides individuals using 16S and ITS2 rRNA gene analyses., Results: Our analyses indicate that archaeal/bacterial and fungal microbiomes varied primarily across broader plant habitat classes (leaves, stems, roots, soils) regardless of plant genotype, except for fungal communities within leaf niches, which were greatly impacted by the host genotype. Differences between tree genotypes are evident in the elevated presence of two potential fungal pathogens, Marssonina brunnea and Septoria sp., on hybrid P. trichocarpa × deltoides trees which may in turn be contributing to divergence in overall microbiome composition. Archaeal/bacterial diversity increased from leaves, to stem, to root, and to soil habitats, whereas fungal diversity was the greatest in stems and soils., Conclusions: This study provides a holistic understanding of microbiome structure within a bioenergy relevant plant host, one of the most complete niche-level analyses of any plant. As such, it constitutes a detailed atlas or map for further hypothesis testing on the significance of individual microbial taxa within specific niches and habitats of Populus and a baseline for comparisons to other plant species.

Background: Bark plays important roles in photosynthate transport and storage, along with physical and chemical protection. Bark texture varies extensively among species, from smooth to fissured to deeply furrowed, but its genetic control is unknown. This study sought to determine the main genomic regions associated with natural variation in bark features and stem diameter. Quantitative trait loci (QTL) were mapped using an interspecific pseudo-backcross pedigree (Populus trichocarpa x P. deltoides and P. deltoides) for bark texture, bark thickness and diameter collected across three years, two sites and three biological replicates per site., Results: QTL specific to bark texture were highly reproducible in shared intervals across sites, years and replicates. Significant positive correlations and co-localization between trait QTL suggest pleiotropic regulators or closely linked genes. A list of candidate genes with related putative function, location close to QTL maxima and with the highest expression level in the phloem, xylem and cambium was identified., Conclusion: Candidate genes for bark texture included an ortholog of Arabidopsis ANAC104 (PopNAC128), which plays a role in lignified fiber cell and ray development, as well as Pinin and Fasciclin (PopFLA) genes with a role in cell adhesion, cell shape and migration. The results presented in this study provide a basis for future genomic characterization of genes found within the QTL for bark texture, bark thickness and diameter in order to better understand stem and bark development in Populus and other woody perennial plants. The QTL mapping approach identified a list of prime candidate genes for further validation using functional genomics or forward genetics approaches.

During symbiosis, organisms use a range of metabolic and protein-based signals to communicate. Of these protein signals, one class is defined as 'effectors', i.e., small secreted proteins (SSPs) that cause phenotypical and physiological changes in another organism. To date, protein-based effectors have been described in aphids, nematodes, fungi and bacteria. Using RNA sequencing of Populus trichocarpa roots in mutualistic symbiosis with the ectomycorrhizal fungus Laccaria bicolor, we sought to determine if host plants also contain genes encoding effector-like proteins. We identified 417 plant-encoded putative SSPs that were significantly regulated during this interaction, including 161 SSPs specific to P. trichocarpa and 15 SSPs exhibiting expansion in Populus and closely related lineages. We demonstrate that a subset of these SSPs can enter L. bicolor hyphae, localize to the nucleus and affect hyphal growth and morphology. We conclude that plants encode proteins that appear to function as effector proteins that may regulate symbiotic associations.

In an effort to better understand the biomass recalcitrance, six natural poplar variants were selected as feedstocks based on previous sugar release analysis. Compositional analysis and physicochemical characterizations of these poplars were performed and the correlations between these physicochemical properties and enzymatic hydrolysis yield were investigated. Gel permeation chromatography (GPC) and 13 C  solid state NMR were used to determine the degree of polymerization (DP) and crystallinity index (CrI) of cellulose, and the results along with the sugar release study indicated that cellulose DP likely played a more important role in enzymatic hydrolysis. Simons' stain revealed that the accessible surface area of substrate significantly varied among these variants from 17.3 to 33.2 mg g-1biomass as reflected by dye adsorption, and cellulose accessibility was shown as one of the major factors governing substrates digestibility. HSQC and 31 P NMR analysis detailed the structural features of poplar lignin variants. Overall, cellulose relevant factors appeared to have a stronger correlation with glucose release, if any, than lignin structural features. Lignin structural features, such as a phenolic hydroxyl group and the ratio of syringyl and guaiacyl (S/G), were found to have a more convincing impact on xylose release. Low lignin content, low cellulose DP, and high cellulose accessibility generally favor enzymatic hydrolysis; however, recalcitrance cannot be simply judged on any single substrate factor., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Plant laccases are thought to function in the oxidation of monolignols which leads to higher order lignin formation. Only a hand-full of laccases in plants have been functionally evaluated, and as such little is known about the breadth of their impact on cell wall chemistry or structure. Here, we describe a previously uncharacterized laccase from Populus, encoded by locus Potri.008G064000, whose reduced expression resulted in transgenic Populus trees with changes in syringyl/guaiacyl ratios as well as altered sugar release phenotypes. These phenotypes are consistent with plant biomass exhibiting reduced recalcitrance. Interestingly, the transgene effect on recalcitrance is dependent on a mild pretreatment prior to chemical extraction of sugars. Metabolite profiling suggests the transgene modulates phenolics that are associated with the cell wall structure. We propose that this particular laccase has a range of functions related to oxidation of phenolics and conjugation of flavonoids that interact with lignin in the cell wall., (© 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Background: Receptor-like kinases (RLKs) belong to a large protein family with over 600 members in Arabidopsis and over 1000 in rice. Among RLKs, the lectin receptor-like kinases (LecRLKs) possess a characteristic extracellular carbohydrate-binding lectin domain and play important roles in plant development and innate immunity. There are 75 and 173 LecRLKs in Arabidopsis and rice, respectively. However, little is known about LecRLKs in perennial woody plants., Results: Here we report the genome-wide analysis of classification, domain architecture and expression of LecRLKs in the perennial woody model plant Populus. We found that the LecRLK family has expanded in Populus to a total of 231, including 180 G-type, 50 L-type and 1 C-type LecRLKs. Expansion of the Populus LecRLKs (PtLecRLKs) occurred partially through tandem duplication. Based on domain architecture and orientation features, we classified PtLecRLKs into eight different classes. RNA-seq-based transcriptomics analysis revealed diverse expression patterns of PtLecRLK genes among leaves, stems, roots, buds and reproductive tissues and organs., Conclusions: This study offers a comprehensive view of LecRLKs in the perennial woody model plant Populus and provides a foundation for functional characterization of this important family of receptor-like kinases.

Strigolactones are a new class of plant hormones regulating shoot branching and symbiotic interactions with arbuscular mycorrhizal fungi. Studies of branching mutants in herbaceous plants have identified several key genes involved in strigolactone biosynthesis or signaling. The strigolactone signal is perceived by a member of the α/β-fold hydrolase superfamily, known as DWARF14 (D14). However, little is known about D14 genes in the woody perennial plants. Here we report the identification of D14 homologs in the model woody plant Populus trichocarpa. We showed that there are two D14 homologs in P. trichocarpa, designated as PtD14a and PtD14b that are over 95% similar at the amino acid level. Expression analysis indicated that the transcript level of PtD14a is generally more abundant than that of PtD14b. However, only PtD14a was able to complement Arabidopsis d14 mutants, suggesting that PtD14a is the functional D14 ortholog. Amino acid alignment and structural modeling revealed substitutions of several highly conserved amino acids in the PtD14b protein including a phenylalanine near the catalytic triad of D14 proteins. This study lays a foundation for further characterization of strigolactone pathway and its functions in the woody perennial plants.

Next-generation sequencing has transformed the ability to link genotypes to phenotypes and facilitates the dissection of genetic contribution to complex traits. However, it is challenging to link genetic variants with the perturbed functional effects on proteins encoded by such genes. Here we show how RNA sequencing can be exploited to construct genotype-specific protein sequence databases to assess natural variation in proteins, providing information about the molecular toolbox driving cellular processes. For this study, we used two natural genotypes selected from a recent genome-wide association study of Populus trichocarpa, an obligate outcrosser with tremendous phenotypic variation across the natural population. This strategy allowed us to comprehensively catalogue proteins containing single amino acid polymorphisms (SAAPs), as well as insertions and deletions. We profiled the frequency of 128 types of naturally occurring amino acid substitutions, including both expected (neutral) and unexpected (non-neutral) SAAPs, with a subset occurring in regions of the genome having strong polymorphism patterns consistent with recent positive and/or divergent selection. By zeroing in on the molecular signatures of these important regions that might have previously been uncharacterized, we now provide a high-resolution molecular inventory that should improve accessibility and subsequent identification of natural protein variants in future genotype-to-phenotype studies.

Background: QTL cloning for the discovery of genes underlying polygenic traits has historically been cumbersome in long-lived perennial plants like Populus. Linkage disequilibrium-based association mapping has been proposed as a cloning tool, and recent advances in high-throughput genotyping and whole-genome resequencing enable marker saturation to levels sufficient for association mapping with no a priori candidate gene selection. Here, multiyear and multienvironment evaluation of cell wall phenotypes was conducted in an interspecific P. trichocarpa x P. deltoides pseudo-backcross mapping pedigree and two partially overlapping populations of unrelated P. trichocarpa genotypes using pyrolysis molecular beam mass spectrometry, saccharification, and/ or traditional wet chemistry. QTL mapping was conducted using a high-density genetic map with 3,568 SNP markers. As a fine-mapping approach, chromosome-wide association mapping targeting a QTL hot-spot on linkage group XIV was performed in the two P. trichocarpa populations. Both populations were genotyped using the 34 K Populus Infinium SNP array and whole-genome resequencing of one of the populations facilitated marker-saturation of candidate intervals for gene identification., Results: Five QTLs ranging in size from 0.6 to 1.8 Mb were mapped on linkage group XIV for lignin content, syringyl to guaiacyl (S/G) ratio, 5- and 6-carbon sugars using the mapping pedigree. Six candidate loci exhibiting significant associations with phenotypes were identified within QTL intervals. These associations were reproducible across multiple environments, two independent genotyping platforms, and different plant growth stages. cDNA sequencing for allelic variants of three of the six loci identified polymorphisms leading to variable length poly glutamine (PolyQ) stretch in a transcription factor annotated as an ANGUSTIFOLIA C-terminus Binding Protein (CtBP) and premature stop codons in a KANADI transcription factor as well as a protein kinase. Results from protoplast transient expression assays suggested that each of the polymorphisms conferred allelic differences in the activation of cellulose, hemicelluloses, and lignin pathway marker genes., Conclusion: This study illustrates the utility of complementary QTL and association mapping as tools for gene discovery with no a priori candidate gene selection. This proof of concept in a perennial organism opens up opportunities for discovery of novel genetic determinants of economically important but complex traits in plants.

Background: UDP-glucose pyrophosphorylase (UGPase) is a sugar-metabolizing enzyme (E.C. 2.7.7.9) that catalyzes a reversible reaction of UDP-glucose and pyrophosphate from glucose-1-phosphate and UTP. UDP-glucose is a key intermediate sugar that is channeled to multiple metabolic pathways. The functional role of UGPase in perennial woody plants is poorly understood., Results: We characterized the functional role of a UGPase gene in Populus deltoides, PdUGPase2. Overexpression of the native gene resulted in increased leaf area and leaf-to-shoot biomass ratio but decreased shoot and root growth. Metabolomic analyses showed that manipulation of PdUGPase2 results in perturbations in primary, as well as secondary metabolism, resulting in reduced sugar and starch levels and increased phenolics, such as caffeoyl and feruloyl conjugates. While cellulose and lignin levels in the cell walls were not significantly altered, the syringyl-to-guaiacyl ratio was significantly reduced., Conclusions: These results demonstrate that PdUGPase2 plays a key role in the tightly coupled primary and secondary metabolic pathways and perturbation in its function results in pronounced effects on growth and metabolism beyond cell wall biosynthesis of Populus.

Forest trees are dominant components of terrestrial ecosystems that have global ecological and economic importance. Despite distributions that span wide environmental gradients, many tree populations are locally adapted, and mechanisms underlying this adaptation are poorly understood. Here we use a combination of whole-genome selection scans and association analyses of 544 Populus trichocarpa trees to reveal genomic bases of adaptive variation across a wide latitudinal range. Three hundred ninety-seven genomic regions showed evidence of recent positive and/or divergent selection and enrichment for associations with adaptive traits that also displayed patterns consistent with natural selection. These regions also provide unexpected insights into the evolutionary dynamics of duplicated genes and their roles in adaptive trait variation.

Background: Strigolactones are a new class of plant hormones that play a key role in regulating shoot branching. Studies of branching mutants in Arabidopsis, pea, rice and petunia have identified several key genes involved in strigolactone biosynthesis or signaling pathway. In the model plant Arabidopsis, MORE AXILLARY GROWTH1 (MAX1), MAX2, MAX3 and MAX4 are four founding members of strigolactone pathway genes. However, little is known about the strigolactone pathway genes in the woody perennial plants., Methodology/principal Finding: Here we report the identification of MAX homologues in the woody model plant Populus trichocarpa. We identified the sequence homologues for each MAX protein in P. trichocarpa. Gene expression analysis revealed that Populus MAX paralogous genes are differentially expressed across various tissues and organs. Furthermore, we showed that Populus MAX genes could complement or partially complement the shoot branching phenotypes of the corresponding Arabidopsis max mutants., Conclusion/significance: This study provides genetic evidence that strigolactone pathway genes are likely conserved in the woody perennial plants and lays a foundation for further characterization of strigolactone pathway and its functions in the woody perennial plants.

In order to uncover the genetic basis of phenotypic trait variation, we used 448 unrelated wild accessions of black cottonwood (Populus trichocarpa) from much of its range in western North America. Extensive data from large-scale trait phenotyping (with spatial and temporal replications within a common garden) and genotyping (with a 34 K Populus single nucleotide polymorphism (SNP) array) of all accessions were used for gene discovery in a genome-wide association study (GWAS). We performed GWAS with 40 biomass, ecophysiology and phenology traits and 29,355 filtered SNPs representing 3518 genes. The association analyses were carried out using a Unified Mixed Model accounting for population structure effects among accessions. We uncovered 410 significant SNPs using a Bonferroni-corrected threshold (P<1.7×10(-6)). Markers were found across 19 chromosomes, explained 1-13% of trait variation, and implicated 275 unique genes in trait associations. Phenology had the largest number of associated genes (240 genes), followed by biomass (53 genes) and ecophysiology traits (25 genes). The GWAS results propose numerous loci for further investigation. Many traits had significant associations with multiple genes, underscoring their genetic complexity. Genes were also identified with multiple trait associations within and/or across trait categories. In some cases, traits were genetically correlated while in others they were not., (© 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.)

Within boreal and temperate forest ecosystems, the majority of trees and shrubs form beneficial relationships with mutualistic ectomycorrhizal (ECM) fungi that support plant health through increased access to nutrients as well as aiding in stress and pest tolerance. The intimate interaction between fungal hyphae and plant roots results in a new symbiotic "organ" called the ECM root tip. Little is understood concerning the metabolic reprogramming that favors the formation of this hybrid tissue in compatible interactions and what prevents the formation of ECM root tips in incompatible interactions. We show here that the metabolic changes during favorable colonization between the ECM fungus Laccaria bicolor and its compatible host, Populus trichocarpa, are characterized by shifts in aromatic acid, organic acid, and fatty acid metabolism. We demonstrate that this extensive metabolic reprogramming is repressed in incompatible interactions and that more defensive compounds are produced or retained. We also demonstrate that L. bicolor can metabolize a number of secreted defensive compounds and that the degradation of some of these compounds produces immune response metabolites (e.g., salicylic acid from salicin). Therefore, our results suggest that the metabolic responsiveness of plant roots to L. bicolor is a determinant factor in fungus-host interactions.

Establishing links between phenotypes and molecular variants is of central importance to accelerate genetic improvement of economically important plant species. Our work represents the first genome-wide association study to the inherently complex and currently poorly understood genetic architecture of industrially relevant wood traits. Here, we employed an Illumina Infinium 34K single nucleotide polymorphism (SNP) genotyping array that generated 29,233 high-quality SNPs in c. 3500 broad-based candidate genes within a population of 334 unrelated Populus trichocarpa individuals to establish genome-wide associations. The analysis revealed 141 significant SNPs (α ≤ 0.05) associated with 16 wood chemistry/ultrastructure traits, individually explaining 3-7% of the phenotypic variance. A large set of associations (41% of all hits) occurred in candidate genes preselected for their suggested a priori involvement with secondary growth. For example, an allelic variant in the FRA8 ortholog explained 21% of the total genetic variance in fiber length, when the trait's heritability estimate was considered. The remaining associations identified SNPs in genes not previously implicated in wood or secondary wall formation. Our findings provide unique insights into wood trait architecture and support efforts for population improvement based on desirable allelic variants., (© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.)

The genetic diversity across natural populations of the model organism, Populus, is extensive, containing a single nucleotide polymorphism roughly every 200 base pairs. When deviations from the reference genome occur in coding regions, they can impact protein sequences. Rather than relying on a static reference database to profile protein expression, we employed a peptide sequence tagging (PST) approach capable of decoding the plasticity of the Populus proteome. Using shotgun proteomics data from two genotypes of P. trichocarpa, a tag-based approach enabled the detection of 6653 unexpected sequence variants. Through manual validation, our study investigated how the most abundant chemical modification (methionine oxidation) could masquerade as a sequence variant (Ala→Ser) when few site-determining ions existed. In fact, precise localization of an oxidation site for peptides with more than one potential placement was indeterminate for 70% of the MS/MS spectra. We demonstrate that additional fragment ions made available by high energy collisional dissociation enhances the robustness of the peptide sequence tagging approach (81% of oxidation events could be exclusively localized to a methionine). We are confident that augmenting fragmentation processes for a PST approach will further improve the identification of single amino acid polymorphism in Populus and potentially other species as well.

Ten years ago, it was announced that the Joint Genome Institute with funds provided by the Department of Energy, Office of Science, Biological and Environmental Research would sequence the black cottonwood (Populus trichocarpa Torr. & Gray) genome. This landmark decision was the culmination of work by the forest science community to develop Populus as a model system. Since its public release in late 2006, the availability of the Populus genome has spawned research in plant biology, morphology, genetics and ecology. Here we address how the tree physiologist has used this resource. More specifically, we revisit our earlier contention that the rewards of sequencing the Populus genome would depend on how quickly scientists working with woody perennials could adopt molecular approaches to investigate the mechanistic underpinnings of basic physiological processes. Several examples illustrate the integration of functional and comparative genomics into the forest sciences, especially in areas that target improved understanding of the developmental differences between woody perennials and herbaceous annuals (e.g., phase transitions). Sequencing the Populus genome and the availability of genetic and genomic resources has also been instrumental in identifying candidate genes that underlie physiological and morphological traits of interest. Genome-enabled research has advanced our understanding of how phenotype and genotype are related and provided insights into the genetic mechanisms whereby woody perennials adapt to environmental stress. In the future, we anticipate that low-cost, high-throughput sequencing will continue to facilitate research in tree physiology and enhance our understanding at scales of individual organisms and populations. A challenge remains, however, as to how genomic resources, including the Populus genome, can be used to understand ecosystem function. Although examples are limited, progress in this area is encouraging and will undoubtedly improve as future research targets the many unique aspects of Populus as a keystone species in terrestrial ecosystems.

Genetic mapping of quantitative traits requires genotypic data for large numbers of markers in many individuals. For such studies, the use of large single nucleotide polymorphism (SNP) genotyping arrays still offers the most cost-effective solution. Herein we report on the design and performance of a SNP genotyping array for Populus trichocarpa (black cottonwood). This genotyping array was designed with SNPs pre-ascertained in 34 wild accessions covering most of the species latitudinal range. We adopted a candidate gene approach to the array design that resulted in the selection of 34 131 SNPs, the majority of which are located in, or within 2 kb of, 3543 candidate genes. A subset of the SNPs on the array (539) was selected based on patterns of variation among the SNP discovery accessions. We show that more than 95% of the loci produce high quality genotypes and that the genotyping error rate for these is likely below 2%. We demonstrate that even among small numbers of samples (n = 10) from local populations over 84% of loci are polymorphic. We also tested the applicability of the array to other species in the genus and found that the number of polymorphic loci decreases rapidly with genetic distance, with the largest numbers detected in other species in section Tacamahaca. Finally, we provide evidence for the utility of the array to address evolutionary questions such as intraspecific studies of genetic differentiation, species assignment and the detection of natural hybrids., (© 2013 Blackwell Publishing Ltd.)

The increasing ecological and economical importance of Populus species and hybrids has stimulated research into the investigation of the natural variation of the species and the estimation of the extent of genetic control over its wood quality traits for traditional forestry activities as well as the emerging bioenergy sector. A realized kinship matrix based on informative, high-density, biallelic single nucleotide polymorphism (SNP) genetic markers was constructed to estimate trait variance components, heritabilities, and genetic and phenotypic correlations. Seventeen traits related to wood chemistry and ultrastructure were examined in 334 9-yr-old Populus trichocarpa grown in a common-garden plot representing populations spanning the latitudinal range 44° to 58.6°. In these individuals, 9342 SNPs that conformed to Hardy-Weinberg expectations were employed to assess the genomic pair-wise kinship to estimate narrow-sense heritabilities and genetic correlations among traits. The range-wide phenotypic variation in all traits was substantial and several trait heritabilities were > 0.6. In total, 61 significant genetic and phenotypic correlations and a network of highly interrelated traits were identified. The high trait variation, the evidence for moderate to high heritabilities and the identification of advantageous trait combinations of industrially important characteristics should aid in providing the foundation for the enhancement of poplar tree breeding strategies for modern industrial use., (© 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.)