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Many industrial chemicals that are produced from fossil resources could be manufactured more sustainably through fermentation. Here we describe the development of a carbon-negative fermentation route to producing the industrially important chemicals acetone and isopropanol from abundant, low-cost waste gas feedstocks, such as industrial emissions and syngas. Using a combinatorial pathway library approach, we first mined a historical industrial strain collection for superior enzymes that we used to engineer the autotrophic acetogen Clostridium autoethanogenum. Next, we used omics analysis, kinetic modeling and cell-free prototyping to optimize flux. Finally, we scaled-up our optimized strains for continuous production at rates of up to ~3 g/L/h and ~90% selectivity. Life cycle analysis confirmed a negative carbon footprint for the products. Unlike traditional production processes, which result in release of greenhouse gases, our process fixes carbon. These results show that engineered acetogens enable sustainable, high-efficiency, high-selectivity chemicals production. We expect that our approach can be readily adapted to a wide range of commodity chemicals.

Background High-throughput metabolomics analytical methodology is needed for population-scale studies of bioenergy-relevant feedstocks such as poplar (Populus sp.). Here, the authors report the relative abundance of secondary aromatic metabolites in Populus trichocarpa leaves rapidly estimated using pyrolysis-molecular beam mass spectrometry (py-MBMS). A training set of leaves was analyzed in conjunction with GC/MS analysis of extracts to determine key spectral features associated with the aromatic metabolites and build PLS models to predict the relative composition of extractable aromatic metabolites in a second test set of leaves. The relative abundance of major secondary aromatic metabolites for the test set of leaves was validated by GC/MS analysis of extracts. Results The Pearson correlation coefficient for the relative abundance of aromatic metabolites based on ranking between GC/MS analysis and py-MBMS analysis of the test leaf set was 0.86 with R2 = 0.76 using a simplified prediction approach from select ions in MBMS spectra. Metabolites most influential to py-MBMS spectral features in the training set included the following compounds: catechol, salicortin, salicyloyl-coumaroyl-glucoside, a-salicyloylsalicin, tremulacin, and to a lesser degree: benzyl-coumaroyl glucoside, benzoyl-salicyloylsalicin, tremuloidin, salireposide, trichocarpin, salicylic acid, salicyl-salicylic acid-2-O-glucoside, phenethyl-tremuloidin, coumaroyl-tremuloidin, 2,6-cyclohexadiene-1,2-diol, and salicyl-tremuloidin. Ions in py-MBMS spectra with the highest correlation to the abundance of aromatic secondary metabolites, particularly those previously mentioned as determined by GC/MS analysis of extracts, included m/z 68, 71, 77, 91, 94, 105, 107, 108, and 122, and were used to develop the simplified prediction approach without PLS models or a priori measurements. Conclusions The simplified py-MBMS method is capable of rapidly screening leaf tissue for relative abundance of extractable aromatic secondary metabolites to enable prioritization of samples in large populations requiring comprehensive metabolomics that will ultimately inform plant systems biology models and advance the development of optimized biomass feedstocks for renewable fuels and chemicals.

The Arabidopsis plastid-localized ALD1 protein acts in the lysine catabolic pathway that produces infection-induced pipecolic acid (Pip), Pip derivatives, and basal non-Pip metabolite(s). ALD1 is indispensable for disease resistance associated with Pseudomonas syringae infections of naïve plants as well as those previously immunized by a local infection, a phenomenon called systemic acquired resistance (SAR). Pseudomonas syringae is known to associate with mesophyll as well as epidermal cells. To probe the importance of epidermal cells in conferring bacterial disease resistance, we studied plants in which ALD1 was only detectable in the epidermal cells of specific leaves. Local disease resistance and many features of SAR were restored when ALD1 preferentially accumulated in the epidermal plastids at immunization sites. Interestingly, SAR restoration occurred without appreciable accumulation of Pip or known Pip derivatives in secondary distal leaves. Our findings establish that ALD1 has a non-autonomous effect on pathogen growth and defense activation. We propose that ALD1 is sufficient in the epidermis of the immunized leaves to activate SAR, but basal ALD1 and possibly a non-Pip metabolite(s) are also needed at all infection sites to fully suppress bacterial growth. Thus, epidermal plastids that contain ALD1 play a key role in local and whole-plant immune signaling.

As the focus for CRISPR/Cas-edited plants moves from proof-of-concept to real-world applications, precise gene manipulation will increasingly require concurrent multiplex editing for polygenic traits. A common approach for editing across multiple sites is to design one guide RNA (gRNA) per target; however, this complicates construct assembly and increases the possibility of off-target mutations. In this study, we utilized one gRNA to target MYB186, a known positive trichome regulator, as well as its paralogs MYB138 and MYB38 at a consensus site for mutagenesis in hybrid poplar (Populus tremula × P. alba INRA 717-1B4). Unexpected duplications of MYB186 and MYB138 resulted in eight alleles for the three targeted genes in the hybrid poplar. Deep sequencing and polymerase chain reaction analyses confirmed editing across all eight targets in nearly all of the resultant glabrous mutants, ranging from small indels to large genomic dropouts, with no off-target activity detected at four potential sites. This highlights the effectiveness of a single gRNA targeting conserved exonic regions for multiplex editing. Additionally, cuticular wax and whole-leaf analyses showed a complete absence of triterpenes in the trichomeless mutants, hinting at a previously undescribed role for the nonglandular trichomes of poplar.

International audience; Trees depend on beneficial interactions between roots and soil microbes for their nutrition and protection against stresses. The soil microbiome provides the main reservoir of microbes for root colonization and is subject to natural variations that can affect its composition. It is not clear whether the tree's root system is able to buffer the natural variations occurring in the soil microbiome to capture a stable and effective microbiome or whether these variations affect its microbiome to impact its physiology. To address this question, we planted cuttings of Gray Poplar (Populus tremula × alba clone 717-1B4) in natural soil taken from a poplar stand under the same tree over two consecutive years and grew them in a greenhouse. We analyzed the soil and root microbiomes by high throughput Illumina MiSeq sequencing of fungal rDNA internal transcribed spacer and bacterial 16S rRNA amplicons and we characterized the root metabolome by gas chromatography-mass spectrometry. Soil and root microbial communities significantly shifted over the 2 years. A modification of the balance between endophytes, saprophytes, and mycorrhizal fungi occurred in the roots as well as a replacement of some dominant operational taxonomic units by others. These modifications were correlated with a significant alteration of the levels of about 10% of primary and secondary metabolites, suggesting that natural fluctuations in soil microbial communities can have a profound impact on tree root metabolism and physiology. Tree roots functioning may thus be indirectly strongly affected by the effects of future extreme climatic variations on the soil microbiome.

Lignin biosynthesis begins with the deamination of phenylalanine and tyrosine (Tyr) as a key branch point between primary and secondary metabolism in land plants. Here, we used a systems biology approach to investigate the global metabolic responses to lignin pathway perturbations in the model grass Brachypodium distachyon. We identified the lignin biosynthetic protein families and found that ammonia-lyases (ALs) are among the most abundant proteins in lignifying tissues in grasses. Integrated metabolomic and proteomic data support a link between lignin biosynthesis and primary metabolism mediated by the ammonia released from ALs that is recycled for the synthesis of amino acids via glutamine. RNA interference knockdown of lignin genes confirmed that the route of the canonical pathway using shikimate ester intermediates is not essential for lignin formation in Brachypodium, and there is an alternative pathway from Tyr via sinapic acid for the synthesis of syringyl lignin involving yet uncharacterized enzymatic steps. Our findings support a model in which plant ALs play a central role in coordinating the allocation of carbon for lignin synthesis and the nitrogen available for plant growth. Collectively, these data also emphasize the value of integrative multiomic analyses to advance our understanding of plant metabolism.

The factors controlling lignin composition remain unclear. Catechyl (C)–lignin is a homopolymer of caffeyl alcohol with unique properties as a biomaterial and precursor of industrial chemicals. The lignin synthesized in the seed coat of Cleome hassleriana switches from guaiacyl (G)– to C-lignin at around 12 to 14 days after pollination (DAP), associated with a rerouting of the monolignol pathway. Lack of synthesis of caffeyl alcohol limits C-lignin formation before around 12 DAP, but coniferyl alcohol is still synthesized and highly accumulated after 14 DAP. We propose a model in which, during C-lignin biosynthesis, caffeyl alcohol noncompetitively inhibits oxidation of coniferyl alcohol by cell wall laccases, a process that might limit movement of coniferyl alcohol to the apoplast. Developmental changes in both substrate availability and laccase specificity together account for the metabolic fates of G- and C-monolignols in the Cleome seed coat.

As the focus for CRISPR edited plants moves from proof-of-concept to real world applications, precise gene manipulation will increasingly require concurrent multiplex editing for polygenic traits. A common approach for editing across multiple sites is to design one gRNA per target; however, this complicates construct assembly and increases the possibility of off-target mutations. In this study, we utilized one gRNA to target MYB186, a known positive trichome regulator, as well as its paralogs MYB138 and MYB38 at a consensus site for mutagenesis in Populus tremula × P. alba INRA 717-1B4. Unexpected duplications of MYB186 and MYB138 resulted in a total of eight alleles for the three targeted genes in the hybrid poplar. Deep sequencing and PCR analyses confirmed editing across all eight targets in nearly all of the resultant glabrous mutants, ranging from small indels to large genomic dropouts, with no off-target activity detected at four potential sites. This highlights the effectiveness of a single gRNA targeting conserved exonic regions for multiplex editing. Additionally, cuticular wax and whole leaf analyses showed a complete absence of triterpenes in the trichomeless mutants, hinting at a previously undescribed role for the non-glandular trichomes of poplar.ONE SENTENCE SUMMARYTargeting conserved sequences with a single gRNA allowed efficient mutagenesis of a multigene family and the recovery of trichomeless and triterpene-free poplar mutants.

A microbe's ecological niche and biotechnological utility are determined by its specific set of co-evolved metabolic pathways. The acquisition of new pathways, through horizontal gene transfer or genetic engineering, can have unpredictable consequences. Here we show that two different pathways for coumarate catabolism failed to function when initially transferred into Escherichia coli. Using laboratory evolution, we elucidated the factors limiting activity of the newly acquired pathways and the modifications required to overcome these limitations. Both pathways required host mutations to enable effective growth with coumarate, but the necessary mutations differed. In one case, a pathway intermediate inhibited purine nucleotide biosynthesis, and this inhibition was relieved by single amino acid replacements in IMP dehydrogenase. A strain that natively contains this coumarate catabolism pathway, Acinetobacter baumannii, is resistant to inhibition by the relevant intermediate, suggesting that natural pathway transfers have faced and overcome similar challenges. Molecular dynamics simulation of the wild type and a representative single-residue mutant provide insight into the structural and dynamic changes that relieve inhibition. These results demonstrate how deleterious interactions can limit pathway transfer, that these interactions can be traced to specific molecular interactions between host and pathway, and how evolution or engineering can alleviate these limitations.

Lignin provides essential mechanical support for plant cell walls but decreases the digestibility of forage crops and increases the recalcitrance of biofuel crops. Attempts to modify lignin content and/or composition by genetic modification often result in negative growth effects. Although several studies have attempted to address the basis for such effects in individual transgenic lines, no common mechanism linking lignin modification with perturbations in plant growth and development has yet been identified. To address whether a common mechanism exists, we have analyzed transposon insertion mutants resulting in independent loss of function of five enzymes of the monolignol pathway, as well as one double mutant, in the model legume Medicago truncatula. These plants exhibit growth phenotypes from essentially wild type to severely retarded. Extensive phenotypic, transcriptomic, and metabolomics analyses, including structural characterization of differentially expressed compounds, revealed diverse phenotypic consequences of lignin pathway perturbation that were perceived early in plant development but were not predicted by lignin content or composition alone. Notable phenotypes among the mutants with severe growth impairment were increased trichome numbers, accumulation of a variety of triterpene saponins, and extensive but differential ectopic expression of defense response genes. No currently proposed model explains the observed phenotypes across all lines. We propose that reallocation of resources into defense pathways is linked to the severity of the final growth phenotype in monolignol pathway mutants of M. truncatula, although it remains unclear whether this is a cause or an effect of the growth impairment.

Serendipitaceae represents a diverse fungal group in the Basidiomycota that includes endophytes and lineages that repeatedly evolved ericoid, orchid and ectomycorrhizal lifestyle. Plants rely upon both nitrogen and phosphorous, for essential growth processes, and are often provided by mycorrhizal fungi. In this study, we investigated the cellular proteome of Serendipita vermifera MAFF305830 and closely related Serendipita vermifera subsp. bescii NFPB0129 grown in vitro under (N) ammonium and (P) phosphate starvation conditions. Mycelial growth pattern was documented under these conditions to correlate growth-specific responses to nutrient starvation. We found that N-starvation accelerated hyphal radial growth, whereas P-starvation accelerated hyphal branching. Additionally, P-starvation triggers an integrated starvation response leading to remodelling of lipid metabolism. Higher abundance of an ammonium transporter known to serve as both an ammonium sensor and stimulator of hyphal growth was detected under N-starvation. Additionally, N-starvation led to strong up-regulation of nitrate, amino acid, peptide, and urea transporters, along with several proteins predicted to have peptidase activity. Taken together, our finding suggests S. bescii and S. vermifera have the metabolic capacity for nitrogen assimilation from organic forms of N compounds. We hypothesize that the nitrogen metabolite repression is a key regulator of such organic N assimilation.

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.

Cell wall recalcitrance is the major obstacle for plant biomass conversion to biofuels. In this study, we functionally characterized a serine hydroxymethyltransferase (SHMT) from Populus and evaluated its potential for developing lignocellulosic feedstocks. SHMT is an enzyme that plays an important role in cellular one-carbon pathways. However, little is known about its function in plant cell wall-related processes. Among nine SHMT genes in the Populus genome, PtSHMT2 was highly expressed in the developing xylem and was co-expressed with secondary cell wall biosynthetic genes. In Populus transgenic plants overexpressing PdSHMT2, the biomass yield and sugar (glucose and xylose) release were increased whereas the lignin content was decreased. Transcriptomics and metabolomics analyses revealed that genes and metabolites related to secondary cell wall biosynthesis were affected by PdSHMT2 overexpression. Based on the transcription factor binding sites of differentially expressed genes in PdSHMT2 overexpression lines, a total of 27 hub transcription factors were identified. We proposed a regulatory model of action of PdSHMT2 with transcriptional master switches of secondary cell wall biosynthesis. Collectively, these results suggest that PdSHMT2 is a promising candidate for genetic engineering to improve feedstock characteristics to enhance biofuel conversion and reduce the cost of lignocellulosic biofuel production.

Pseudomonas syringae is a ubiquitous plant pathogen, infecting both woody and herbaceous plants and resulting in devastating agricultural crop losses. Characterized by a remarkable specificity for plant hosts, P. syringae pathovars utilize a number of virulence factors including the type III secretion system and effector proteins to elicit disease in a particular host species. Here, two Pseudomonas syringae strains were isolated from diseased Populustrichocarpa seeds. The pathovars were capable of inhibiting poplar seed germination and were selective for the Populus genus. Sequencing of the newly described organisms revealed similarity to phylogroup II pathogens and genomic regions associated with woody host-associated plant pathogens, as well as genes for specific virulence factors. The host response to infection, as revealed through metabolomics, is the induction of the stress response through the accumulation of higher-order salicylates. Combined with necrosis on leaf surfaces, the plant appears to quickly respond by isolating infected tissues and mounting an anti-inflammatory defense. This study improves our understanding of the initial host response to epiphytic pathogens in Populus and provides a new model system for studying the effects of a bacterial pathogen on a woody host plant in which both organisms are fully genetically sequenced.

Climate change causes significant alterations in precipitation and temperature regimes that are predicted to become more extreme throughout the next century. Microorganisms are important members within ecosystems, and how they respond to these changing abiotic stressors has large implications for the functioning of ecosystems, the recycling of nutrients, and the health of the aboveground plant community. Drought stress negatively impacts microbial activity, but the magnitude of this stress response may be dependent on above- and belowground interactions. This study demonstrates that beneficial associations between plants and microbes can enhance tolerance to abiotic stress., Drought stress negatively impacts microbial activity, but the magnitude of stress responses is likely dependent on a diversity of belowground interactions. Populus trichocarpa individuals and no-plant bulk soils were exposed to extended drought (∼0.03% gravimetric water content [GWC] after 12 days), rewet, and a 12-day “recovery” period to determine the effects of plant presence in mediating soil microbiome stability to water stress. Plant metabolomic analyses indicated that drought exposure increased host investment in C and N metabolic pathways (amino acids, fatty acids, phenolic glycosides) regardless of recovery. Several metabolites positively correlated with root-associated microbial alpha-diversity, but not those of soil communities. Soil bacterial community composition shifted with P. trichocarpa presence and with drought relative to irrigated controls, whereas soil fungal composition shifted only with plant presence. However, root fungal communities strongly shifted with drought, whereas root bacterial communities changed to a lesser degree. The proportion of bacterial water-stress opportunistic operational taxonomic units (OTUs) (enriched counts in drought) was high (∼11%) at the end of drying phases and maintained after rewet and recovery phases in bulk soils, but it declined over time in soils with plants present. For root fungi, opportunistic OTUs were high at the end of recovery in drought treatments (∼17% abundance), although relatively not responsive in soils, particularly planted soils (

Gas fermentation by autotrophic bacteria, such as clostridia, offers a sustainable path to numerous bioproducts from a range of local, highly abundant, waste and low-cost feedstocks, such as industrial flue gases or syngas generated from biomass or municipal waste. Unfortunately, designing and engineering clostridia remains laborious and slow. The ability to prototype individual genetic parts, gene expression, and biosynthetic pathway performance in vitro before implementing them in cells could help address these bottlenecks by speeding up design. Unfortunately, a high-yielding cell-free gene expression (CFE) system from clostridia has yet to be developed. Here, we report the development and optimization of a high-yielding (236 ± 24 µg/mL) batch CFE platform from the industrially relevant anaerobe, Clostridium autoethanogenum. A key feature of the platform is that both circular and linear DNA templates can be applied directly to the CFE reaction to program protein synthesis. We demonstrate the ability to prototype gene expression, and quantitatively map cell-free metabolism in lysates from this system. We anticipate that the C. autoethanogenum CFE platform will not only expand the protein synthesis toolkit for synthetic biology, but also serve as a platform in expediting the screening and prototyping of gene regulatory elements in non-model, industrially relevant microbes.

Summary 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.

Summary The apparent antagonism between salicylic acid (SA) and jasmonic acid (JA)/ethylene (ET) signalling resulting in trade‐offs between defence against (hemi)biotrophic and necrotrophic pathogens has been widely described across multiple plant species. However, the underlying mechanism remains to be fully established.The molecular and cellular functions of ANGUSTIFOLIA (AN) were characterised, and its role in regulating the pathogenic response was studied in Arabidopsis.We demonstrated that AN, a plant homologue of mammalian C‐TERMINAL BINDING PROTEIN (CtBP), antagonistically regulates plant resistance to the hemibiotrophic pathogen Pseudomonas syringae and the necrotrophic pathogen Botrytis cinerea. Consistent with phenotypic observations, transcription of genes involved in SA and JA/ET pathways was antagonistically regulated by AN. By interacting with another nuclear protein TYROSYL‐DNA PHOSPHODIESTERASE1 (TDP1), AN imposes transcriptional repression on MYB46, encoding a transcriptional activator of PHENYLALANINE AMMONIA‐LYASE (PAL) genes which are required for SA biosynthesis, while releasing TDP1‐imposed transcriptional repression on WRKY33, a master regulator of the JA/ET signalling pathway.These findings demonstrate that transcriptional co‐regulation of MYB46 and WRKY33 by AN mediates the coordination of SA and JA/ET pathways to optimise defences against (hemi)biotrophic and necrotrophic pathogens.

In plants, the phenylpropanoid pathway is responsible for the synthesis of a diverse array of secondary metabolites that include lignin monomers, flavonoids, and coumarins, many of which are essential for plant structure, biomass recalcitrance, stress defense, and nutritional quality. Our previous studies have demonstrated that Populus trichocarpa PtrEPSP‐TF, an isoform of 5‐enolpyruvylshikimate 3‐phosphate (EPSP) synthase, has transcriptional activity and regulates phenylpropanoid biosynthesis in Populus. In this study, we report the identification of single nucleotide polymorphism (SNP) of PtrEPSP‐TF that defines its functionality. Populus natural variants carrying this SNP were shown to have reduced lignin content. Here, we demonstrated that the SNP‐induced substitution of 142nd amino acid (PtrEPSP‐TFD142E) dramatically impairs the DNA‐binding and transcriptional activity of PtrEPSP‐TF. When introduced to a monocot species rice (Oryza sativa) in which an EPSP synthase isoform with the DNA‐binding helix‐turn‐helix (HTH) motif is absent, the PtrEPSP‐TF, but not PtrEPSP‐TFD142E, activated genes in the phenylpropanoid pathway. More importantly, heterologous expression of PtrEPSP‐TF uncovered five new transcriptional regulators of phenylpropanoid biosynthesis in rice. Collectively, this study identifies the key amino acid required for PtrEPSP‐TF functionality and provides a strategy to uncover new transcriptional regulators in phenylpropanoid biosynthesis.

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.

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.

We examined the effect of downregulating PdKOR1 gene, an endo-β-1,4-glucanase gene family member previously characterized to affect cellulose biosynthesis and cell wall composition in Populus, on the secondary metabolome and microbiome of field-grown Populus deltoides. We revealed differences in metabolite profiles of PdKOR1 RNAi and control roots using gas chromatography-mass spectrometry, and microbiome identification via Illumina MiSeq 16S and ITS2 rRNA sequencing in root endospheres and rhizospheres. PdKOR1 RNAi root metabolites differed from control plants: free amino acids (valine, isoleucine, alanine) were reduced while caffeoyl-shikimates, salicylic-acid derivatives, and flavonoid metabolites increased in PdKOR1 RNAi roots. The Actinobacterial family Micromonosporaceae were more abundant in RNAi root endospheres, whereas Nitrospirae was reduced in PdKOR1 RNAi rhizospheres. Ascomycota were lower and Basidiomycota greater in PdKOR1 rhizospheres. Bacterial and fungal community composition, as measured by Bray-Curtis dissimilarity, differed between PdKOR1 RNAi and control rhizospheres and endospheres. These results indicate that modification of plant cell walls via downregulation of PdKOR1 gene in Populus impacts carbon metabolism in roots and concomitant alterations in root-associated microbial communities. Such an understanding of functional and ecological implications of biomass chemistry improvement efforts is critical to address the goals of sustainable bioenergy crop production and management.

An amendment to this paper has been published and can be accessed via the original article.

The black cottonwood poplar (Populus trichocarpa) leaf ionome (inorganic trace elements and mineral nutrients) is an important aspect for determining the physiological and developmental processes contributing to biomass production. A number of techniques are used to measure the ionome, yet characterizing the leaf spatial heterogeneity remains a challenge, especially in solid samples. L aser- i nduced b reakdown s pectroscopy (LIBS) has been used to determine the elemental composition of leaves and is able to raster across solid matrixes at 10 μm resolution. Here, we evaluate the use of LIBS for solid sample leaf elemental characterization in relation to neutron activation. In fact, neutron activation analysis is a laboratory-based technique which is used by the National Institute of Standards and Technology (NIST) to certify trace elements in candidate reference materials including plant leaf matrices. Introduction to the techniques used in this research has been presented in this manuscript. N eutron a ctivation a nalysis (NAA) data has been correlated to the LIBS spectra to achieve quantification of the elements or ions present within poplar leaves. The regression coefficients of calibration and validation using m ulti v ariate a nalysis (MVA) methodology for six out of seven elements have been determined and vary between 0.810 and 0.998. LIBS and NAA data has been presented for the elements such as, calcium, magnesium, manganese, aluminum, copper, and potassium. Chlorine was also detected but it did not show good correlation between the LIBS and NAA techniques. This research shows that LIBS can be used as a fast, high-spatial resolution technique to quantify elements as part of large-scale field phenotyping projects.

We examined variation in growth rate, patterns of nitrogen utilization, and competitive interactions of Atractiella rhizophila isolates from the roots of Populus hosts. Atractiella grew significantly faster on media substituted with inorganic nitrogen sources and slower in the presence of another fungal genus. To determine plausible causal mechanisms we used metabolomics to explore competitive interactions between Atractiella strains and Fusarium oxysporum or Leptosphaerulina chartarum. Metabolomic screening of potential microbial inhibitors showed increased levels of glycosides produced in vitro by Atractiella when grown with a different fungal genus, relative to when grown alone. Cumulatively, these results suggest Atractiella is a poor competitor with other fungi via direct routes e.g. faster growth rates, but may utilize chemical interactions and possibly nitrogen sources to defend itself, and niche partition its way to abundance in the plant host root and rhizosphere.

Populus has been investigated as a promising biomass feedstock for alternative fuels and chemicals. Physicochemical characteristics and genomic information of biomass feedstocks are among the essential information that can help not only advance our understanding of biomass recalcitrance but also in its efficient utilization. Herein, the recalcitrance of natural Populus variants was elucidated in three aspects: (1) sugar release, (2) physicochemical properties, and (3) relative variation of gene expression within natural poplar variants. The sugar release performance of natural Populus variants was evaluated with their correlation with biomass recalcitrance-related characteristics. Among the physicochemical properties of poplar, the lignin content, lignin molecular weight, lignin S/G ratio, and cellulose accessibility were found to correlate with sugar release. The results demonstrated that the lignin content was negatively correlated with sugar release, whereas the lignin molecular weight, lignin S/G ratio, and cellulose accessibility were positively associated with poplar sugar release. The trend of differential gene expression of each variant also supports the characterization results and their effects on biomass conversion.

Lignin provides essential mechanical support for plant cell walls but decreases the digestibility of forage crops and increases the recalcitrance of biofuel crops. Attempts to modify lignin content and/or composition by genetic modification often result in negative growth effects. Although several studies have attempted to address the basis for such effects in individual transgenic lines, no common mechanism linking lignin modification with perturbations in plant growth and development has yet been identified. To address whether a common mechanism exists, we have analyzed transposon insertion mutants resulting in independent loss of function of five enzymes of the monolignol pathway, as well as one double mutant, in the model legume Medicago truncatula. These plants exhibit growth phenotypes from essentially wild type to severely retarded. Extensive phenotypic, transcriptomic, and metabolomics analyses, including structural characterization of differentially expressed compounds, revealed diverse phenotypic consequences of lignin pathway perturbation that were perceived early in plant development but were not predicted by lignin content or composition alone. Notable phenotypes among the mutants with severe growth impairment were increased trichome numbers, accumulation of a variety of triterpene saponins, and extensive but differential ectopic expression of defense response genes. No currently proposed model explains the observed phenotypes across all lines. We propose that reallocation of resources into defense pathways is linked to the severity of the final growth phenotype in monolignol pathway mutants of M. truncatula, although it remains unclear whether this is a cause or an effect of the growth impairment.