Metatranscriptomics sheds light on the links between the functional traits of fungal guilds and ecological processes in forest soil ecosystems.

Soil fungi belonging to different functional guilds, such as saprotrophs, pathogens, and mycorrhizal symbionts, play key roles in forest ecosystems. To date, no study has compared the actual gene expression of these guilds in different forest soils. We used metatranscriptomics to study the competition for organic resources by these fungal groups in boreal, temperate, and Mediterranean forest soils. Using a dedicated mRNA annotation pipeline combined with the JGI MycoCosm database, we compared the transcripts of these three fungal guilds, targeting enzymes involved in C- and N mobilization from plant and microbial cell walls. Genes encoding enzymes involved in the degradation of plant cell walls were expressed at a higher level in saprotrophic fungi than in ectomycorrhizal and pathogenic fungi. However, ectomycorrhizal and saprotrophic fungi showed similarly high expression levels of genes encoding enzymes involved in fungal cell wall degradation. Transcripts for N-related transporters were more highly expressed in ectomycorrhizal fungi than in other groups. We showed that ectomycorrhizal and saprotrophic fungi compete for N in soil organic matter, suggesting that their interactions could decelerate C cycling. Metatranscriptomics provides a unique tool to test controversial ecological hypotheses and to better understand the underlying ecological processes involved in soil functioning and carbon stabilization.

Soil fauna-microbial interactions shifts fungal and bacterial communities under a contamination disturbance.

The aim of this study was to determine whether the soil faunal-microbial interaction complexity (SFMIC) is a significant factor influencing the soil microbial communities and the willow growth in the context of PAH contamination. The SFMIC treatment had eight levels: just the microbial community, or the microbial community with nematodes, springtails, earthworms and all the possible combinations. SFMIC affected the height and biomass of willows after eight weeks or growth. SFMIC affected the structure and the composition of the bacterial, archaeal and fungal communities, with significant effects of SFMIC on the relative abundance of fungal genera such as Sphaerosporella, a known willow symbiont during phytoremediation, and bacterial phyla such as Actinobacteriota, containing many polycyclic aromatic hydrocarbons (PAH) degraders. These SFMIC effects on microbial communities were not clearly reflected in the community structure and abundance of PAH degraders, even though some degraders related to Actinobacteriota and the diversity of Gram-negative degraders were affected by the SFMIC treatments. Over 95% of PAH was degraded in all pots at the end of the experiment. Overall, our results suggest that, under our experimental conditions, SFMIC changes willow phytoremediation outcomes.

Overexpression of REDUCED WALL ACETYLATION C increases xylan acetylation and biomass recalcitrance in Populus.

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.

Microbial community structural and functional differentiation in capped thickened oil sands tailings planted with native boreal species.

The oil sands mining operations in Alberta have produced billions of m3 of tailings which must be reclaimed and integrated into various mine closure landforms, including terrestrial landforms. Microorganisms play a central role in nutrient cycling during the reclamation of disturbed landscapes, contributing to successful vegetation restoration and long-term sustainability. However, microbial community succession and response in reconstructed and revegetated tailings remain largely unexplored. This study aimed to monitor the structural and functional responses of microbial communities in tailings subjected to different capping and vegetation strategies over two growing seasons (GS). To achieve this, a column-based greenhouse experiment was conducted to investigate microbial communities in tailings that were capped with a layer (10 or 30 cm) of peat-mineral mix (PMM) and planted with either upland or wetland communities. DNA metabarcoding analysis of the bacterial 16S rRNA gene and fungal ITS2 region as well as shotgun metagenomics were used to asses the impact of treatments on microbial taxonomy and functions, respectively. Results showed that tailings microbial diversity and community composition changed considerably after two GS compared to baseline samples, while communities in the PMM capping layer were much more stable. Likewise, several microbial functions were significantly enriched in tailings after two GS. Interestingly, the impact of capping on bacterial communities in tailings varied depending on the plant community, leading to a higher number of differentially abundant taxa and to a decrease in Shannon diversity and evenness in the upland treatment but not in the wetland treatment. Moreover, while capping in the presence of wetland vegetation increased the energy-related metabolic functions (carbon, nitrogen, and sulfur), these functions were depleted by capping in the upland treatment. Fungi represented a small proportion of the microbial community in tailings, but the relative abundance of several taxa changed over time, while the capping treatments favored the growth of some beneficial taxa, notably the root endophyte Serendipita, in both upland and wetland columns. The results suggest that selecting the right combination of capping material and vegetation type may contribute to improve below-ground microbial processes and sustain plant growth in harsh environments such as oil sands tailings.

Does wood mulch trigger microbially mediated positive plant-soil feedback in degraded boreal forest sites? A post hoc study.

Introduction: Reforestation of degraded lands in the boreal forest is challenging and depends on the direction and strength of the plant-soil feedback (PSF). Methods: Using a gradient in tree productivity (null, low and high) from a long-term, spatially replicated reforestation experiment of borrow pits in the boreal forest, we investigated the interplay between microbial communities and soil and tree nutrient stocks and concentrations in relation to a positive PSF induced by wood mulch amendment. Results: Three levels of mulch amendment underlie the observed gradient in tree productivity, and plots that had been amended with a continuous layer of mulch 17 years earlier showed a positive PSF with trees up to 6 m tall, a closed canopy, and a developing humus layer. The average taxonomic and functional composition of the bacterial and fungal communities differed markedly betweenlow- and high-productivity plots. Trees in high-productivity plots recruited a specialized soil microbiome that was more efficient at nutrient mobilization and acquisition. These plots showed increases in carbon (C), calcium (Ca), nitrogen (N), potassium (K), and phosphorus (P) stocks and as well as bacterial and fungal biomass. The soil microbiome was dominated by taxa from the fungal genus Cortinarius and the bacterial family Chitinophagaceae, and a complex microbial network with higher connectivity and more keystone species supported tree productivity in reforested plots compared to unproductive plots. Discussion: Therefore, mulching of plots resulted in a microbially mediated PSF that enhances mineral weathering and non-symbiotic N fixation, and in turn helps transform unproductive plots into productive plots to ensure rapid restoration of the forest ecosystem in a harsh boreal environment.

Integrative analysis of green ash phloem transcripts and proteins during an emerald ash borer infestation.

BACKGROUND: Emerald ash borer (Agrilus planipennis; EAB) is an Asian insect species that has been invasive to North America for 20 years. During this time, the emerald ash borer has killed tens of millions of American ash (Fraxinus spp) trees. Understanding the inherent defenses of susceptible American ash trees will provide information to breed new resistant varieties of ash trees. RESULTS: We have performed RNA-seq on naturally infested green ash (F. pennsylvanica) trees at low, medium and high levels of increasing EAB infestation and proteomics on low and high levels of EAB infestation. Most significant transcript changes we detected occurred between the comparison of medium and high levels of EAB infestation, indicating that the tree is not responding to EAB until it is highly infested. Our integrative analysis of the RNA-Seq and proteomics data identified 14 proteins and 4 transcripts that contribute most to the difference between highly infested and low infested trees. CONCLUSIONS: The putative functions of these transcripts and proteins suggests roles of phenylpropanoid biosynthesis and oxidation, chitinase activity, pectinesterase activity, strigolactone signaling, and protein turnover.

Understanding Willow Transcriptional Response in the Context of Oil Sands Tailings Reclamation.

One of the reclamation objectives for treated oil sands tailings (OST) is to establish boreal forest communities that can integrate with the surrounding area. Hence, selection of appropriate soil reclamation cover designs and plant species for revegetation are important aspects of tailings landform reclamation and closure. Research and monitoring of the long term and immediate impacts of capped OST on the growth and survival of native boreal plant species are currently underway. However, plant responses to OST-associated toxicity are not well known at the molecular level. Using RNA sequencing, we examined the effects of three types of OST on the willow transcriptome under different capping strategies. The transcriptomic data showed that some genes respond universally and others in a specific manner to different types of OST. Among the dominant and shared upregulated genes, we found some encoding protein detoxification (PD), Cytochrome P450 (CYPs), glutathione S-transferase regulatory process (GST), UDP-glycosyltransferase (UGT), and ABC transporter and regulatory process associated proteins. Moreover, genes encoding several stress-responsive transcription factors (bZIP, BHLH, ERF, MYB, NAC, WRKY) were upregulated with OST-exposure, while high numbers of transcripts related to photosynthetic activity and chloroplast structure and function were downregulated. Overall, the expression of 40 genes was found consistent across all tailings types and capping strategies. The qPCR analysis of a subset of these shared genes suggested that they could reliably distinguish plants exposed to different OST associated stress. Our results indicated that it is possible to develop OST stress exposure biosensors merely based on changes in the level of expression of a relatively small set of genes. The outcomes of this study will further guide optimization of OST capping and revegetation technology by using knowledge based plant stress adaptation strategies.

Unrelated Fungal Rust Candidate Effectors Act on Overlapping Plant Functions.

Rust fungi cause epidemics that threaten the production of important plant species, such as wheat and soy. Melampsora larici-populina (Mlp) causes the poplar rust and encodes at least 1184 candidate effectors (CEs) whose functions are poorly known. In this study, we sequenced the transcriptome and used mass spectrometry to analyze the metabolome of Arabidopsis plants constitutively expressing 14 Mlp CEs and of a control line to discover alterations leading to plant susceptibility. We found 2299 deregulated genes across the experiment. Genes involved in pattern-triggered immunity, such as FRK1, PR1, RBOHD, and WRKY33, as well as AUX/IAA genes were down-regulated. We further observed that 680 metabolites were deregulated in at least one CE-expressing transgenic line, with "highly unsaturated and phenolic compounds" and "peptides" enriched among down- and up-regulated metabolites. Interestingly, transgenic lines expressing unrelated CEs had correlated patterns of gene and metabolite deregulation, while expression of CEs belonging to the same family deregulated different genes and metabolites. Thus, our results uncouple effector sequence similarity and function. This supports that effector functional investigation in the context of their virulence activity and effect on plant susceptibility requires the investigation of the individual effector and precludes generalization based on sequence similarity.

Nitrogen isotopes in the soil-to-tree continuum - Tree rings express the soil biogeochemistry of boreal forests exposed to moderate airborne emissions.

Anthropogenic N emissions represent a potential threat for forest ecosystems, and environmental indicators that provide insight into the changing forest N cycle are needed. Tree ring N isotopic ratios (δ15N) appear as a contentious choice for this role as the exact mechanisms behind tree-ring δ15N changes seldom benefit from a scrutiny of the soil-to-tree N continuum. This study integrates the results from the analysis of soil chemistry, soil microbiome genomics, and δ15N values of soil N compounds, roots, ectomycorrhizal (EcM) fungi and recent tree rings of thirteen white spruce trees sampled in five stands, from two regions exposed to moderate anthropogenic N emissions (3.9 to 8.1 kg/ha/y) with distinctive δ15N signals. Our results reveal that airborne anthropogenic N with distinct δ15N signals may directly modify the NO3- δ15N values in surface soils, but not the ones of NH4+, the preferred N form of the studied trees. Hence, the tree-ring δ15N values reflect specific soil N conditions and assimilation modes by trees. Along with a wide tree-ring δ15N range, we report differences in: soil nutrient content and N transformation rates; δ15N values of NH4+, total dissolved N (TDN) and EcM mantle enveloping the root tips; and bacterial and fungal community structures. We combine EcM mantle and root δ15N values with fungal identification to infer that hydrophobic EcM fungi transfer N from the dissolved organic N (DON) pool to roots under acidic conditions, and hydrophilic EcM fungi transfer various N forms to roots, which also assimilate N directly under less acidic conditions. Despite the complexities of soil biogeochemical properties and processes identified in the studied sites, in the end, the tree-ring δ15N averages inversely correlate with soil pH and anthropogenic N inputs, confirming white spruce tree-ring δ15N values as a suitable indicator for environmental research on forest N cycling.

Soil Characteristics Constrain the Response of Microbial Communities and Associated Hydrocarbon Degradation Genes during Phytoremediation.

Rhizodegradation is a promising cleanup technology where microorganisms degrade soil contaminants in the rhizosphere. A symbiotic relationship is expected to occur between plant roots and soil microorganisms in contaminated soils that enhances natural microbial degradation. However, little is known about how different initial microbiotas influence the rhizodegradation outcome. Recent studies have hinted that soil initial diversity has a determining effect on the outcome of contaminant degradation. To test this, we either planted (P) or not (NP) balsam poplars (Populus balsamifera) in two soils of contrasting diversity (agricultural and forest) that were contaminated or not with 50 mg kg-1 of phenanthrene (PHE). The DNA from the rhizosphere of the P and the bulk soil of the NP pots was extracted and the bacterial genes encoding the 16S rRNA, the PAH ring-hydroxylating dioxygenase alpha subunits (PAH-RHDα) of Gram-positive and Gram-negative bacteria, and the fungal ITS region were sequenced to characterize the microbial communities. The abundances of the PAH-RHDα genes were quantified by real-time quantitative PCR. Plant presence had a significant effect on PHE degradation only in the forest soil, whereas both NP and P agricultural soils degraded the same amount of PHE. Fungal communities were mainly affected by plant presence, whereas bacterial communities were principally affected by the soil type, and upon contamination the dominant PAH-degrading community was similarly constrained by soil type. Our results highlight the crucial importance of soil microbial and physicochemical characteristics in the outcome of rhizoremediation.IMPORTANCE Polycyclic aromatic hydrocarbons (PAH) are a group of organic contaminants that pose a risk to ecosystems' health. Phytoremediation is a promising biotechnology with the potential to restore PAH-contaminated soils. However, some limitations prevent it from becoming the remediation technology of reference, despite being environmentally friendlier than mainstream physicochemical alternatives. Recent reports suggest that the original soil microbial diversity is the key to harnessing the potential of phytoremediation. Therefore, this study focused on determining the effect of two different soil types in the fate of phenanthrene (a polycyclic aromatic hydrocarbon) under balsam poplar remediation. Poplar increased the degradation of phenanthrene in forest, but not in agricultural soil. The fungi were affected by poplars, whereas total bacteria and specific PAH-degrading bacteria were constrained by soil type, leading to different degradation patterns between soils. These results highlight the importance of performing preliminary microbiological studies of contaminated soils to determine whether plant presence could improve remediation rates or not.

Plant Genotype Influences Physicochemical Properties of Substrate as Well as Bacterial and Fungal Assemblages in the Rhizosphere of Balsam Poplar.

Abandoned unrestored mines are an important environmental concern as they typically remain unvegetated for decades, exposing vast amounts of mine waste to erosion. Several factors limit the revegetation of these sites, including extreme abiotic and unfavorable biotic conditions. However, some pioneer tree species having high levels of genetic diversity, such as balsam poplar (Populus balsamifera), can naturally colonize these sites and initiate plant succession. This suggests that some tree genotypes are likely more suited for acclimation to the conditions of mine wastes. In this study, we selected two contrasting mine waste storage facilities (waste rock from a gold mine and tailings from a molybdenum mine) from the Abitibi region of Quebec (Canada), on which poplars were found to have grown naturally. First, we assessed in situ the impact of vegetation presence on each mine waste type. The presence of balsam poplars improved soil health locally by modifying the physicochemical properties (e.g., higher nutrient content and pH) of the mine wastes and causing an important shift in their bacterial and fungal community compositions, going from lithotrophic communities that dominate mine waste environments to heterotrophic communities involved in nutrient cycling. Next, in a greenhouse experiment we assessed the impact of plant genotype when grown in these mine wastes. Ten genotypes of P. balsamifera were collected locally, found growing either at the mine sites or in the surrounding natural forest. Tree growth was monitored over two growing seasons, after which the effects of genotype-by-environment interactions were assessed by measuring the physicochemical properties of the substrates and the changes in microbial community assembly. Although substrate type was identified as the main driver of rhizosphere microbiome diversity and community structure, a significant effect due to tree genotype was also detected, particularly for bacterial communities. Plant genotype also influenced aboveground tree growth and the physicochemical properties of the substrates. These results highlight the influence of balsam poplar genotype on the soil environment and the potential importance of tree genotype selection in the context of mine waste revegetation.

The Ecobiomics project: Advancing metagenomics assessment of soil health and freshwater quality in Canada.

Transformative advances in metagenomics are providing an unprecedented ability to characterize the enormous diversity of microorganisms and invertebrates sustaining soil health and water quality. These advances are enabling a better recognition of the ecological linkages between soil and water, and the biodiversity exchanges between these two reservoirs. They are also providing new perspectives for understanding microorganisms and invertebrates as part of interacting communities (i.e. microbiomes and zoobiomes), and considering plants, animals, and humans as holobionts comprised of their own cells as well as diverse microorganisms and invertebrates often acquired from soil and water. The Government of Canada's Genomics Research and Development Initiative (GRDI) launched the Ecobiomics Project to coordinate metagenomics capacity building across federal departments, and to apply metagenomics to better characterize microbial and invertebrate biodiversity for advancing environmental assessment, monitoring, and remediation activities. The Project has adopted standard methods for soil, water, and invertebrate sampling, collection and provenance of metadata, and nucleic acid extraction. High-throughput sequencing is located at a centralized sequencing facility. A centralized Bioinformatics Platform was established to enable a novel government-wide approach to harmonize metagenomics data collection, storage and bioinformatics analyses. Sixteen research projects were initiated under Soil Microbiome, Aquatic Microbiome, and Invertebrate Zoobiome Themes. Genomic observatories were established at long-term environmental monitoring sites for providing more comprehensive biodiversity reference points to assess environmental change.

Variations in terrestrial arthropod DNA metabarcoding methods recovers robust beta diversity but variable richness and site indicators.

Terrestrial arthropod fauna have been suggested as a key indicator of ecological integrity in forest systems. Because phenotypic identification is expert-limited, a shift towards DNA metabarcoding could improve scalability and democratize the use of forest floor arthropods for biomonitoring applications. The objective of this study was to establish the level of field sampling and DNA extraction replication needed for arthropod biodiversity assessments from soil. Processing 15 individually collected soil samples recovered significantly higher median richness (488-614 sequence variants) than pooling the same number of samples (165-191 sequence variants) prior to DNA extraction, and we found no significant richness differences when using 1 or 3 pooled DNA extractions. Beta diversity was robust to changes in methodological regimes. Though our ability to identify taxa to species rank was limited, we were able to use arthropod COI metabarcodes from forest soil to assess richness, distinguish among sites, and recover site indicators based on unnamed exact sequence variants. Our results highlight the need to continue DNA barcoding local taxa during COI metabarcoding studies to help build reference databases. All together, these sampling considerations support the use of soil arthropod COI metabarcoding as a scalable method for biomonitoring.

Functional Analysis of the PgCesA3 White Spruce Cellulose Synthase Gene Promoter in Secondary Xylem.

Cellulose is an essential structural component of the plant cell wall. Its biosynthesis involves genes encoding cellulose synthase enzymes and a complex transcriptional regulatory network. Three cellulose synthases have been identified in conifers as being potentially involved in secondary cell wall biosynthesis because of their preferential expression in xylem tissues; however, no direct functional association has been made to date. In the present work, we characterized the white spruce [Picea glauca (Moench) Voss] cellulose synthase PgCesA3 gene and 5' regulatory elements. Phylogenetic analysis showed that PgCesA1-3 genes grouped with secondary cell wall-associated Arabidopsis cellulose synthase genes, such as AtCesA8, AtCesA4, and AtCesA7. We produced transgenic spruce expressing the GUS reporter gene driven by the PgCesA3 promoter. We observed blue staining in differentiating xylem cells from stem and roots, and in foliar guard cells indicating that PgCesA3 is clearly involved in secondary cell wall biosynthesis. The promoter region sequence of PgCesA3 contained several putative MYB cis-regulatory elements including AC-I like motifs and secondary wall MYB-responsive element (SMRE); however, it lacked SMRE4, 7 and 8 that correspond to the sequences of AC-I, II, and III. Based on these findings and results of previous transient trans-activation assays that identified interactions between the PgCesA3 promoter and different MYB transcription factors, we performed electrophoretic mobility shift assays with MYB recombinant proteins and cis-regulatory elements present in the PgCesA3 promoter. We found that PgMYB12 bound to a canonical AC-I element identified in the Pinus taeda PAL promoter and two AC-I like elements. We hypothesized that the PgMYB12 could regulate PgCesA3 in roots based on previous expression results. This functional study of PgCesA3 sequences and promoter opens the door for future studies on the interaction between PgMYBs and the PgCesA3 regulatory elements.

Rhizoremediation of petroleum hydrocarbons: a model system for plant microbiome manipulation.

Phytoremediation is a green and sustainable alternative to physico-chemical methods for contaminated soil remediation. One of the flavours of phytoremediation is rhizoremediation, where plant roots stimulate soil microbes to degrade organic contaminants. This approach is particularly interesting as it takes advantage of naturally evolved interaction mechanisms between plant and microorganisms and often results in a complete mineralization of the contaminants (i.e. transformation to water and CO2 ). However, many biotic and abiotic factors influence the outcome of this interaction, resulting in variable efficiency of the remediation process. The difficulty to predict precisely the timeframe associated with rhizoremediation leads to low adoption rates of this green technology. Here, we review recent literature related to rhizoremediation, with a particular focus on soil organisms. We then expand on the potential of rhizoremediation to be a model plant-microbe interaction system for microbiome manipulation studies.

The impact of reconstructed soils following oil sands exploitation on aspen and its associated belowground microbiome.

The objective of this study was to investigate the impact of different soil covers used to reclaim decommissioned oil sands mining sites on the genetic diversity of aspen and their associated belowground microbiota. Aspen genotyping showed that trees mostly originated from sexual reproduction on sites reclaimed with soil covers made of upland forest floor-mineral mix (FFMM) and lowland peat-mineral mix (PMM). In contrast, most individuals in mature and burned stands sampled as benchmarks for natural disturbances originated from vegetative reproduction. Nonetheless, aspen populations in the FFMM and PMM sites were not genetically different from those in mature and burned stands. DNA metabarcoding of bacteria and fungi in root and soil samples revealed that the diversity of the belowground microbiota associated with aspen and the relative abundance of putative symbiotic taxa in PMM were significantly lower than for FFMM and naturally disturbed sites. Despite similar aspen genetic diversity between FFMM and PMM sites, trees were not associated with the same belowground microbiota. Because the soil microbiome and more specifically the mycorrhizal communities are variable both in space and time, long-term monitoring is particularly important to better understand the ecological trajectory of these novel ecosystems.

Infection assays in Arabidopsis reveal candidate effectors from the poplar rust fungus that promote susceptibility to bacteria and oomycete pathogens.

Fungi of the Pucciniales order cause rust diseases which, altogether, affect thousands of plant species worldwide and pose a major threat to several crops. How rust effectors-virulence proteins delivered into infected tissues to modulate host functions-contribute to pathogen virulence remains poorly understood. Melampsora larici-populina is a devastating and widespread rust pathogen of poplar, and its genome encodes 1184 identified small secreted proteins that could potentially act as effectors. Here, following specific criteria, we selected 16 candidate effector proteins and characterized their virulence activities and subcellular localizations in the leaf cells of Arabidopsis thaliana. Infection assays using bacterial (Pseudomonas syringae) and oomycete (Hyaloperonospora arabidopsidis) pathogens revealed subsets of candidate effectors that enhanced or decreased pathogen leaf colonization. Confocal imaging of green fluorescent protein-tagged candidate effectors constitutively expressed in stable transgenic plants revealed that some protein fusions specifically accumulate in nuclei, chloroplasts, plasmodesmata and punctate cytosolic structures. Altogether, our analysis suggests that rust fungal candidate effectors target distinct cellular components in host cells to promote parasitic growth.

In vivo function of Pgßglu-1 in the release of acetophenones in white spruce.

Eastern spruce budworm (Choristoneura fumiferiana Clemens) (ESBW) is a major forest pest which feeds on young shoots of white spruce (Picea glauca) and can cause landscape level economic and ecological losses. Release of acetophenone metabolites, piceol and pungenol, from their corresponding glycosides, picein and pungenin, can confer natural resistance of spruce to ESBW. A beta-glucosidase gene, Pgßglu-1, was recently discovered and the encoded enzyme was characterized in vitro to function in the release of the defensive acetophenone aglycons. Here we describe overexpression of Pgßglu-1 in a white spruce genotype whose metabolome contains the glucosylated acetophenones, but no detectable amounts of the aglycons. Transgenic overexpression of Pgßglu-1 resulted in release of the acetophenone aglycons in planta. This work provides in vivo evidence for the function of Pgßglu-1.

Poplar MYB115 and MYB134 Transcription Factors Regulate Proanthocyanidin Synthesis and Structure.

The accumulation of proanthocyanidins is regulated by a complex of transcription factors composed of R2R3 MYB, basic helix-loop-helix, and WD40 proteins that activate the promoters of biosynthetic genes. In poplar (genus Populus), MYB134 is known to regulate proanthocyanidin biosynthesis by activating key flavonoid genes. Here, we characterize a second MYB regulator of proanthocyanidins, MYB115. Transgenic poplar overexpressing MYB115 showed a high-proanthocyanidin phenotype and reduced salicinoid accumulation, similar to the effects of MYB134 overexpression. Transcriptomic analysis of MYB115- and MYB134-overexpressing poplar plants identified a set of common up-regulated genes encoding proanthocyanidin biosynthetic enzymes and several novel uncharacterized MYB transcriptional repressors. Transient expression experiments demonstrated the capacity of both MYB134 and MYB115 to activate flavonoid promoters, but only in the presence of a basic helix-loop-helix cofactor. Yeast two-hybrid experiments confirmed the direct interaction of these transcription factors. The unexpected identification of dihydromyricetin in leaf extracts of both MYB115- and MYB134-overexpressing poplar led to the discovery of enhanced flavonoid B-ring hydroxylation and an increased proportion of prodelphinidins in proanthocyanidin of the transgenics. The dramatic hydroxylation phenotype of MYB115 overexpressors is likely due to the up-regulation of both flavonoid 3',5'-hydroxylases and cytochrome b5 Overall, this work provides new insight into the complexity of the gene regulatory network for proanthocyanidin synthesis in poplar.