Learning from methylomes: epigenomic correlates of Populus balsamifera traits based on deep learning models of natural DNA methylation.

Epigenomes have remarkable potential for the estimation of plant traits. This study tested the hypothesis that natural variation in DNA methylation can be used to estimate industrially important traits in a genetically diverse population of Populus balsamifera L. (balsam poplar) trees grown at two common garden sites. Statistical learning experiments enabled by deep learning models revealed that plant traits in novel genotypes can be modelled transparently using small numbers of methylated DNA predictors. Using this approach, tissue type, a nonheritable attribute, from which DNA methylomes were derived was assigned, and provenance, a purely heritable trait and an element of population structure, was determined. Significant proportions of phenotypic variance in quantitative wood traits, including total biomass (57.5%), wood density (40.9%), soluble lignin (25.3%) and cell wall carbohydrate (mannose: 44.8%) contents, were also explained from natural variation in DNA methylation. Modelling plant traits using DNA methylation can capture tissue-specific epigenetic mechanisms underlying plant phenotypes in natural environments. DNA methylation-based models offer new insight into natural epigenetic influence on plants and can be used as a strategy to validate the identity, provenance or quality of agroforestry products.

Sexual epigenetics: gender-specific methylation of a gene in the sex determining region of Populus balsamifera.

Methylation has frequently been implicated in gender determination in plants. The recent discovery of the sex determining region (SDR) of balsam poplar, Populus balsamifera, pinpointed 13 genes with differentiated X and Y copies. We tested these genes for differential methylation using whole methylome sequencing of xylem tissue of multiple individuals grown under field conditions in two common gardens. The only SDR gene to show a marked pattern of gender-specific methylation is PbRR9, a member of the two component response regulator (type-A) gene family, involved in cytokinin signalling. It is an ortholog of Arabidopsis genes ARR16 and ARR17. The strongest patterns of differential methylation (mostly male-biased) are found in the putative promoter and the first intron. The 4th intron is strongly methylated in both sexes and the 5th intron is unmethylated in both sexes. Using a statistical learning algorithm we find that it is possible accurately to assign trees to gender using genome-wide methylation patterns alone. The strongest predictor is the region coincident with PbRR9, showing that this gene stands out against all genes in the genome in having the strongest sex-specific methylation pattern. We propose the hypothesis that PbRR9 has a direct, epigenetically mediated, role in poplar sex determination.

Leaf size serves as a proxy for xylem vulnerability to cavitation in plantation trees.

Hybrid poplars are an important renewable forest resource known for their high productivity. At the same time, they are highly vulnerable to water stress. Identifying traits that can serve as indicators for growth performance remains an important task, particularly under field conditions. Understanding which trait combinations translate to improved productivity is key in order to satisfy the demand for poplar wood in an uncertain future climate. In this study, we compared hydraulic and leaf traits among five hybrid poplar clones at 10 plantations in central Alberta. We also assessed the variation of these traits between 2- to 3-year-old branches from the lower to mid-crown and current-year long shoots from the mid to upper crown. Our results showed that (1) hybrid poplars differed in key hydraulic parameters between branch type, (2) variation of hydraulic traits among clones was relatively large for some clones and less for others, and (3) strong relationships between measured hydraulic traits, such as vessel diameter, cavitation resistance, xylem-specific and leaf-specific conductivity and leaf area, were observed. Our results suggest that leaf size could serve as an additional screening tool when selecting for drought-tolerant genotypes in forest management and tree improvement programmes.

Comprehensive multiphase NMR: a promising technology to study plants in their native state.

Nuclear magnetic resonance (NMR) spectroscopy is arguably one the most powerful tools to study the interactions and molecular structure within plants. Traditionally, however, NMR has developed as two separate fields, one dealing with liquids and the other dealing with solids. Plants in their native state contain components that are soluble, swollen, and true solids. Here, a new form of NMR spectroscopy, developed in 2012, termed comprehensive multiphase (CMP)-NMR is applied for plant analysis. The technology composes all aspects of solution, gel, and solid-state NMR into a single NMR probe such that all components in all phases in native unaltered samples can be studied and differentiated in situ. The technology is evaluated using wild-type Arabidopsis thaliana and the cellulose-deficient mutant ectopic lignification1 (eli1) as examples. Using CMP-NMR to study intact samples eliminated the bias introduced by extraction methods and enabled the acquisition of a more complete structural and metabolic profile; thus, CMP-NMR revealed molecular differences between wild type (WT) and eli1 that could be overlooked by conventional methods. Methanol, fatty acids and/or lipids, glutamine, phenylalanine, starch, and nucleic acids were more abundant in eli1 than in WT. Pentaglycine was present in A. thaliana seedlings and more abundant in eli1 than in WT.

Poplar trees reconfigure the transcriptome and metabolome in response to drought in a genotype- and time-of-day-dependent manner.

BACKGROUND: Drought has a major impact on tree growth and survival. Understanding tree responses to this stress can have important application in both conservation of forest health, and in production forestry. Trees of the genus Populus provide an excellent opportunity to explore the mechanistic underpinnings of forest tree drought responses, given the growing molecular resources that are available for this taxon. Here, foliar tissue of six water-deficit stressed P. balsamifera genotypes was analysed for variation in the metabolome in response to drought and time of day by using an untargeted metabolite profiling technique, gas chromatography/mass-spectrometry (GC/MS). RESULTS: Significant variation in the metabolome was observed in response the imposition of water-deficit stress. Notably, organic acid intermediates such as succinic and malic acid had lower concentrations in leaves exposed to drought, whereas galactinol and raffinose were found in increased concentrations. A number of metabolites with significant difference in accumulation under water-deficit conditions exhibited intraspecific variation in metabolite accumulation. Large magnitude fold-change accumulation was observed in three of the six genotypes. In order to understand the interaction between the transcriptome and metabolome, an integrated analysis of the drought-responsive transcriptome and the metabolome was performed. One P. balsamifera genotype, AP-1006, demonstrated a lack of congruence between the magnitude of the drought transcriptome response and the magnitude of the metabolome response. More specifically, metabolite profiles in AP-1006 demonstrated the smallest changes in response to water-deficit conditions. CONCLUSIONS: Pathway analysis of the transcriptome and metabolome revealed specific genotypic responses with respect to primary sugar accumulation, citric acid metabolism, and raffinose family oligosaccharide biosynthesis. The intraspecific variation in the molecular strategies that underpin the responses to drought among genotypes may have an important role in the maintenance of forest health and productivity.

Investigating the drought-stress response of hybrid poplar genotypes by metabolite profiling.

Drought stress is perhaps the most commonly encountered abiotic stress plants experience in the natural environment, and it is one of the most important factors limiting plant productivity. Here, we employed untargeted metabolite profiling to examine four drought-stressed hybrid poplar (Populus spp.) genotypes for their metabolite content, using gas chromatography coupled to mass spectrometry. The primary objective of these analyses was to characterize the metabolite profile of poplar trees to assess relative drought resistance and to investigate the underlying biochemical mechanisms employed by the genotypes to combat drought. Metabolite profiling identified key metabolites that increased or decreased in relative abundance upon exposure to drought stress. Overall, amino acids, the antioxidant phenolic compounds catechin and kaempferol, as well as the osmolytes raffinose and galactinol exhibited increased abundance under drought stress, whereas metabolites involved in photorespiration, redox regulation and carbon fixation showed decreased abundance under drought stress. One clone in particular, Okanese, displayed unique responses to the imposed drought conditions. This clone was found to have higher leaf water potential, but lower growth rate relative to the other clones tested. Okanese also had lower accumulation of osmolytes such as raffinose, galactinol and proline, but higher overall levels of antioxidants such as catechin and dehydroascorbic acid. As such, it was proposed that osmotic adjustment as a mechanism for drought avoidance in this clone is not as well developed in comparison with the other clones investigated in this study, and that a possible alternative mechanism for the enhanced drought avoidance displayed by Okanese may be due to differential allocation of resources or better retention of water.

Interactions between the R2R3-MYB transcription factor, AtMYB61, and target DNA binding sites.

Despite the prominent roles played by R2R3-MYB transcription factors in the regulation of plant gene expression, little is known about the details of how these proteins interact with their DNA targets. For example, while Arabidopsis thaliana R2R3-MYB protein AtMYB61 is known to alter transcript abundance of a specific set of target genes, little is known about the specific DNA sequences to which AtMYB61 binds. To address this gap in knowledge, DNA sequences bound by AtMYB61 were identified using cyclic amplification and selection of targets (CASTing). The DNA targets identified using this approach corresponded to AC elements, sequences enriched in adenosine and cytosine nucleotides. The preferred target sequence that bound with the greatest affinity to AtMYB61 recombinant protein was ACCTAC, the AC-I element. Mutational analyses based on the AC-I element showed that ACC nucleotides in the AC-I element served as the core recognition motif, critical for AtMYB61 binding. Molecular modelling predicted interactions between AtMYB61 amino acid residues and corresponding nucleotides in the DNA targets. The affinity between AtMYB61 and specific target DNA sequences did not correlate with AtMYB61-driven transcriptional activation with each of the target sequences. CASTing-selected motifs were found in the regulatory regions of genes previously shown to be regulated by AtMYB61. Taken together, these findings are consistent with the hypothesis that AtMYB61 regulates transcription from specific cis-acting AC elements in vivo. The results shed light on the specifics of DNA binding by an important family of plant-specific transcriptional regulators.

Interplay between sucrose and folate modulates auxin signaling in Arabidopsis.

As sessile organisms growing in an ever-changing environment, plants must integrate multiple regulatory inputs to promote the appropriate developmental responses. One such nutritional signal is cellular sugar levels, which rise and fall throughout the day and affect a variety of developmental processes. To uncover signaling pathways that modulate sugar perception, compounds from the Library of Active Compounds in Arabidopsis were screened for the ability to perturb developmental responses to sucrose (Suc) in Arabidopsis (Arabidopsis thaliana) seedlings. This screen found that sulfonamides, which inhibit folate biosynthesis in plants, restrict hypocotyl elongation in a sugar-dependent fashion. Transcriptome analysis identified a small set of transcripts that respond to the interaction between sulfonamide and Suc, including a number of transcripts encoding Auxin/Indole-3-Acetic Acids, negative regulators of auxin signal transduction. Chemical inhibition of auxin transport or genetic disruption of auxin signaling relieved this interaction, suggesting that responses to these two nutritional stimuli are mediated by auxin. Reporter systems used to track auxin signaling and distribution showed enhanced activity in the vascular region of the hypocotyl in response to cotreatment of Suc and sulfonamide, yet no change in auxin abundance was observed. Taken together, these findings suggest that the interplay between Suc and folates acts to fine-tune auxin sensitivity and influences auxin distribution during seedling development.

Epigenetic regulation of adaptive responses of forest tree species to the environment.

Epigenetic variation is likely to contribute to the phenotypic plasticity and adaptative capacity of plant species, and may be especially important for long-lived organisms with complex life cycles, including forest trees. Diverse environmental stresses and hybridization/polyploidization events can create reversible heritable epigenetic marks that can be transmitted to subsequent generations as a form of molecular "memory". Epigenetic changes might also contribute to the ability of plants to colonize or persist in variable environments. In this review, we provide an overview of recent data on epigenetic mechanisms involved in developmental processes and responses to environmental cues in plant, with a focus on forest tree species. We consider the possible role of forest tree epigenetics as a new source of adaptive traits in plant breeding, biotechnology, and ecosystem conservation under rapid climate change.

Constitutive expression of a fungal glucuronoyl esterase in Arabidopsis reveals altered cell wall composition and structure.

A family 15 carbohydrate esterase (CE15) from the white-rot basidiomycete, Phanerochaete carnosa (PcGCE), was transformed into Arabidopsis thaliana Col-0 and was expressed from the constitutive cauliflower mosaic virus 35S promoter. Like other CE15 enzymes, PcGCE hydrolyzed methyl-4-O-methyl-d-glucopyranuronate and could target ester linkages that contribute to lignin-carbohydrate complexes that form in plant cell walls. Three independently transformed Arabidopsis lines were evaluated in terms of nine morphometric parameters, total sugar and lignin composition, cell wall anatomy, enzymatic saccharification and xylan extractability. The transgenic lines consistently displayed a leaf-yellowing phenotype, as well as reduced glucose and xylose content by as much as 30% and 35%, respectively. Histological analysis revealed 50% reduction in cell wall thickness in the interfascicular fibres of transgenic plants, and FT-IR microspectroscopy of interfascicular fibre walls indicated reduction in lignin cross-linking in plants overexpressing PcGCE. Notably, these characteristics could be correlated with improved xylose recovery in transgenic plants, up to 15%. The current analysis represents the first example whereby a fungal glucuronoyl esterase is expressed in Arabidopsis and shows that the promotion of glucuronoyl esterase activity in plants can alter the extent of intermolecular cross-linking within plant cell walls.

Drought induces alterations in the stomatal development program in Populus.

Much is known about the physiological control of stomatal aperture as a means by which plants adjust to water availability. By contrast, the role played by the modulation of stomatal development to limit water loss has received much less attention. The control of stomatal development in response to water deprivation in the genus Populus is explored here. Drought induced declines in stomatal conductance as well as an alteration in stomatal development in two genotypes of Populus balsamifera. Leaves that developed under water-deficit conditions had lower stomatal indices than leaves that developed under well-watered conditions. Transcript abundance of genes that could hypothetically underpin drought-responsive changes in stomatal development was examined, in two genotypes, across six time points, under two conditions, well-watered and with water deficit. Populus homologues of STOMAGEN, ERECTA (ER), STOMATA DENSITY AND DISTRIBUTION 1 (SDD1), and FAMA had variable transcript abundance patterns congruent with their role in the modulation of stomatal development in response to drought. Conversely, there was no significant variation in transcript abundance between genotypes or treatments for the Populus homologues of YODA (YDA) and TOO MANY MOUTHS (TMM). The findings highlight the role that could be played by stomatal development during leaf expansion as a longer term means by which to limit water loss from leaves. Moreover, the results point to the key roles played by the regulation of the homologues of STOMAGEN, ER, SDD1, and FAMA in the control of this response in poplar.

AtMYB61, an R2R3-MYB transcription factor, functions as a pleiotropic regulator via a small gene network.

Throughout their lifetimes, plants must coordinate the regulation of various facets of growth and development. Previous evidence has suggested that the Arabidopsis thaliana R2R3-MYB, AtMYB61, might function as a coordinate regulator of multiple aspects of plant resource allocation. Using a combination of cell biology, transcriptome analysis and biochemistry, in conjunction with gain-of-function and loss-of-function genetics, the role of AtMYB61 in conditioning resource allocation throughout the plant life cycle was explored. In keeping with its role as a regulator of resource allocation, AtMYB61 is expressed in sink tissues, notably xylem, roots and developing seeds. Loss of AtMYB61 function decreases xylem formation, induces qualitative changes in xylem cell structure and decreases lateral root formation; in contrast, gain of AtMYB61 function has the opposite effect on these traits. AtMYB61 coordinates a small network of downstream target genes, which contain a motif in their upstream regulatory regions that is bound by AtMYB61, and AtMYB61 activates transcription from this same motif. Loss-of-function analysis supports the hypothesis that AtMYB61 targets play roles in shaping subsets of AtMYB61-related phenotypes. Taken together, these findings suggest that AtMYB61 links the transcriptional control of multiple aspects of plant resource allocation.

The interaction between MYB proteins and their target DNA binding sites.

Members of the MYB family of transcription factors are found in all eukaryotic lineages, where they function to regulate either fundamental cellular processes, or specific facets of metabolism or cellular differentiation. MYB transcription factors regulate these processes through modulation of transcription at target genes, to which they bind in a sequence-specific manner. Over the past decades, insights have been gained into the molecular interactions between MYB proteins and their cognate DNA targets. This review focuses on those insights, the emergence of common themes in DNA binding by diverse MYB family members. The review also considers gaps in the current knowledge of MYB-DNA interactions, particularly for plant MYB proteins, and how emerging techniques that examine protein-DNA interactions can fill these gaps.

Clone history shapes Populus drought responses.

Just as animal monozygotic twins can experience different environmental conditions by being reared apart, individual genetically identical trees of the genus Populus can also be exposed to contrasting environmental conditions by being grown in different locations. As such, clonally propagated Populus trees provide an opportunity to interrogate the impact of individual environmental history on current response to environmental stimuli. To test the hypothesis that current responses to an environmental stimulus, drought, are contingent on environmental history, the transcriptome- level drought responses of three economically important hybrid genotypes-DN34 (Populus deltoides × Populus nigra), Walker [P. deltoides var. occidentalis × (Populus laurifolia × P. nigra)], and Okanese [Walker × (P. laurifolia × P. nigra)]-derived from two different locations were compared. Strikingly, differences in transcript abundance patterns in response to drought were based on differences in geographic origin of clones for two of the three genotypes. This observation was most pronounced for the genotypes with the longest time since establishment and last common propagation. Differences in genome-wide DNA methylation paralleled the transcriptome level trends, whereby the clones with the most divergent transcriptomes and clone history had the most marked differences in the extent of total DNA methylation, suggesting an epigenomic basis for the clone history-dependent transcriptome divergence. The data provide insights into the interplay between genotype and environment in the ecologically and economically important Populus genus, with implications for the industrial application of Populus trees and the evolution and persistence of these important tree species and their associated hybrids.

Transcriptomic responses of the softwood-degrading white-rot fungus Phanerochaete carnosa during growth on coniferous and deciduous wood.

To identify enzymes that could be developed to reduce the recalcitrance of softwood resources, the transcriptomes of the softwood-degrading white-rot fungus Phanerochaete carnosa were evaluated after growth on lodgepole pine, white spruce, balsam fir, and sugar maple and compared to the transcriptome of P. carnosa after growth on liquid nutrient medium. One hundred fifty-two million paired-end reads were obtained, and 63% of these reads were mapped to 10,257 gene models from P. carnosa. Five-hundred thirty-three of these genes had transcripts that were at least four times more abundant during growth on at least one wood medium than on nutrient medium. The 30 transcripts that were on average over 100 times more abundant during growth on wood than on nutrient medium included 6 manganese peroxidases, 5 cellulases, 2 hemicellulases, a lignin peroxidase, glyoxal oxidase, and a P450 monooxygenase. Notably, among the genes encoding putative cellulases, one encoding a glycosyl hydrolase family 61 protein had the highest relative transcript abundance during growth on wood. Overall, transcripts predicted to encode lignin-degrading activities were more abundant than those predicted to encode carbohydrate-active enzymes. Transcripts predicted to encode three MnPs represented the most highly abundant transcripts in wood-grown cultivations compared to nutrient medium cultivations. Gene set enrichment analyses did not distinguish transcriptomes resulting from softwood and hardwood cultivations, suggesting that similar sets of enzyme activities are elicited by P. carnosa grown on different wood substrates, albeit to different expression levels.

PlaNet: combined sequence and expression comparisons across plant networks derived from seven species.

The model organism Arabidopsis thaliana is readily used in basic research due to resource availability and relative speed of data acquisition. A major goal is to transfer acquired knowledge from Arabidopsis to crop species. However, the identification of functional equivalents of well-characterized Arabidopsis genes in other plants is a nontrivial task. It is well documented that transcriptionally coordinated genes tend to be functionally related and that such relationships may be conserved across different species and even kingdoms. To exploit such relationships, we constructed whole-genome coexpression networks for Arabidopsis and six important plant crop species. The interactive networks, clustered using the HCCA algorithm, are provided under the banner PlaNet (http://aranet.mpimp-golm.mpg.de). We implemented a comparative network algorithm that estimates similarities between network structures. Thus, the platform can be used to swiftly infer similar coexpressed network vicinities within and across species and can predict the identity of functional homologs. We exemplify this using the PSA-D and chalcone synthase-related gene networks. Finally, we assessed how ontology terms are transcriptionally connected in the seven species and provide the corresponding MapMan term coexpression networks. The data support the contention that this platform will considerably improve transfer of knowledge generated in Arabidopsis to valuable crop species.

Endogenous overexpression of Populus MYB186 increases trichome density, improves insect pest resistance, and impacts plant growth.

Trichomes are specialized epidermal cells that generally play a role in reducing transpiration and act as a deterrent to herbivory. In a screen of activation-tagged Populus tremula × Populus alba 717-1B4 trees, we identified a mutant line, fuzzy, with increased foliar trichome density. This mutant also had a 35% increase in growth rate and a 200% increase in the rate of photosynthesis as compared with wild-type poplar. The fuzzy mutant had significant resistance to feeding by larvae of the white-spotted tussock moth (Orgyia leucostigma), a generalist insect pest of poplar trees. The fuzzy trichome phenotype is attributable to activation tagging and increased expression of the gene encoding PtaMYB186, which is related to Arabidopsis thaliana MYB106, a known regulator of trichome initiation. The fuzzy phenotype can be recapitulated by overexpressing PtaMYB186 in poplar. PtaMYB186 overexpression results in reconfiguration of the poplar transcriptome, with changes in the transcript abundance of suites of genes that are related to trichome differentiation. It is notable that a plant with misexpression of a gene responsible for trichome development also had altered traits related to growth rate and pest resistance, suggesting that non-intuitive facets of plant development might be useful targets for plant improvement.

Genome-wide analysis of plant metal transporters, with an emphasis on poplar.

The specific transport of metal ions, mediated by membrane-localized metal transporters, is of fundamental importance in all eukaryotes. Genome-wide analysis of metal transporters was undertaken, making use of whole genome sequences of the green alga Chlamydomonas reinhardtii, the moss Physcomitrella patens, the lycophyte Selaginella moellendorffii, the monocots rice and sorghum, and the dicots Arabidopsis thaliana, poplar, grapevine, as well as of the yeast Saccharomyces cerevisiae. A repertoire of 430 metal transporters was found in total across eight photosynthetic plants, as well as in S. cerevisiae. Seventy-two full-length metal transporter genes were identified in the Populus genome alone, which is the largest number of metal transporters genes identified in any single species to date. Diversification of some transporter family gene clusters appears to have occurred in a lineage-specific manner. Expression analysis of Populus metal transporters indicates that some family members show tissue-specific transcript abundance. Taken together, the data provide a picture into the diversification of these important gene families.

Intraspecific variation in the Populus balsamifera drought transcriptome.

Drought is a major limitation to the growth and productivity of trees in the ecologically and economically important genus Populus. The ability of Populus trees to contend with drought is a function of genome responsiveness to this environmental insult, involving reconfiguration of the transcriptome to appropriately remodel growth, development and metabolism. Here we test hypotheses aimed at examining the extent of intraspecific variation in the drought transcriptome using six different Populus balsamifera L. genotypes and Affymetrix GeneChip technology. Within a given genotype there was a positive correlation between the magnitude of water-deficit induced changes in transcript abundance across the transcriptome, and the capacity of that genotype to maintain growth following water deficit. Genotypes that had more similar drought-responsive transcriptomes also had fewer genotypic differences, as determined by microarray-derived single feature polymorphism (SFP) analysis, suggesting that responses may be conserved across individuals that share a greater degree of genotypic similarity. This work highlights the fact that a core species-level response can be defined; however, the underpinning genotype-derived complexities of the drought response in Populus must be taken into consideration when defining both species- and genus-level responses.

Time of day shapes Arabidopsis drought transcriptomes.

Under natural conditions, it is common for plants to experience water deprivation (drought) for periods of days or longer. Plants respond to drought stress by reconfiguring their transcriptome activity. Transcriptome changes in response to drought are dynamic, and are shaped by mitigating factors like time during the diurnal cycle. To date, analyses of drought-induced transcriptome remodelling have concentrated on dynamic changes induced by rapid desiccation, or changes at a single time point following gradual water stress. To gain insights into the dynamics of transcriptome reconfiguration in response to gradual drying of the soil, the drought-induced transcriptomes of Arabidopsis thaliana were examined at four time points over a single diel period - midday, late day, midnight, and pre-dawn. Transcriptome reconfigurations were induced by drought in advance of changes to relative water content, leaf water loss, and chlorophyll content. Comparative analyses support the hypothesis that the drought-responsive transcriptomes were shaped by invocation of distinct hormonal and stress response pathways at different times of the day. While a core set of genes were drought responsive at multiple time points throughout the day, the magnitude of the response varied in a manner dependent on the time of day. Moreover, analysis of a single time point would fail to identify suites of drought-responsive genes that can only be detected through assessment of the dynamics of diurnal changes, emphasising the value of characterising multiple time-of-day-specific drought transcriptomes.