Proteomic and metabolic disturbances in lignin-modified Brachypodium distachyon.

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.

Ectomycorrhizal symbiosis prepares its host locally and systemically for abiotic cue signaling.

Tree growth and survival are dependent on their ability to perceive signals, integrate them, and trigger timely and fitted molecular and growth responses. While ectomycorrhizal symbiosis is a predominant tree-microbe interaction in forest ecosystems, little is known about how and to what extent it helps trees cope with environmental changes. We hypothesized that the presence of Laccaria bicolor influences abiotic cue perception by Populus trichocarpa and the ensuing signaling cascade. We submitted ectomycorrhizal or non-ectomycorrhizal P. trichocarpa cuttings to short-term cessation of watering or ozone fumigation to focus on signaling networks before the onset of any physiological damage. Poplar gene expression, metabolite levels, and hormone levels were measured in several organs (roots, leaves, mycorrhizas) and integrated into networks. We discriminated the signal responses modified or maintained by ectomycorrhization. Ectomycorrhizas buffered hormonal changes in response to short-term environmental variations systemically prepared the root system for further fungal colonization and alleviated part of the root abscisic acid (ABA) signaling. The presence of ectomycorrhizas in the roots also modified the leaf multi-omics landscape and ozone responses, most likely through rewiring of the molecular drivers of photosynthesis and the calcium signaling pathway. In conclusion, P. trichocarpa-L. bicolor symbiosis results in a systemic remodeling of the host's signaling networks in response to abiotic changes. In addition, ectomycorrhizal, hormonal, metabolic, and transcriptomic blueprints are maintained in response to abiotic cues, suggesting that ectomycorrhizas are less responsive than non-mycorrhizal roots to abiotic challenges.

Expression quantitative trait loci mapping identified PtrXB38 as a key hub gene in adventitious root development in Populus.

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

Multiplex knockout of trichome-regulating MYB duplicates in hybrid poplar using a single gRNA.

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.

Towards engineering ectomycorrhization into switchgrass bioenergy crops via a lectin receptor-like kinase.

Soil-borne microbes can establish compatible relationships with host plants, providing a large variety of nutritive and protective compounds in exchange for photosynthesized sugars. However, the molecular mechanisms mediating the establishment of these beneficial relationships remain unclear. Our previous genetic mapping and whole-genome resequencing studies identified a gene deletion event of a Populus trichocarpa lectin receptor-like kinase gene PtLecRLK1 in Populus deltoides that was associated with poor-root colonization by the ectomycorrhizal fungus Laccaria bicolor. By introducing PtLecRLK1 into a perennial grass known to be a non-host of L. bicolor, switchgrass (Panicum virgatum L.), we found that L. bicolor colonizes ZmUbipro-PtLecRLK1 transgenic switchgrass roots, which illustrates that the introduction of PtLecRLK1 has the potential to convert a non-host to a host of L. bicolor. Furthermore, transcriptomic and proteomic analyses on inoculated-transgenic switchgrass roots revealed genes/proteins overrepresented in the compatible interaction and underrepresented in the pathogenic defence pathway, consistent with the view that pathogenic defence response is down-regulated during compatible interaction. Metabolomic profiling revealed that root colonization in the transgenic switchgrass was associated with an increase in N-containing metabolites and a decrease in organic acids, sugars, and aromatic hydroxycinnamate conjugates, which are often seen in the early steps of establishing compatible interactions. These studies illustrate that PtLecRLK1 is able to render a plant susceptible to colonization by the ectomycorrhizal fungus L. bicolor and shed light on engineering mycorrhizal symbiosis into a non-host to enhance plant productivity and fitness on marginal lands.

ALD1 accumulation in Arabidopsis epidermal plastids confers local and non-autonomous disease resistance.

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.

A 5-Enolpyruvylshikimate 3-Phosphate Synthase Functions as a Transcriptional Repressor in Populus.

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.

Horizontal transfer of a pathway for coumarate catabolism unexpectedly inhibits purine nucleotide biosynthesis.

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.

Overexpression of a Prefoldin ß subunit gene reduces biomass recalcitrance in the bioenergy crop Populus.

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.

Development of a clostridia-based cell-free system for prototyping genetic parts and metabolic pathways.

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 part function, gene expression patterns, and biosynthetic pathway performance in vitro before implementing designs 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 aerobic 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.

Arabidopsis C-terminal binding protein ANGUSTIFOLIA modulates transcriptional co-regulation of MYB46 and WRKY33.

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.

Ectopic Defense Gene Expression Is Associated with Growth Defects in Medicago truncatula Lignin Pathway Mutants.

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.

Underground Azelaic Acid-Conferred Resistance to Pseudomonas syringae in Arabidopsis.

Local interactions between individual plant organs and diverse microorganisms can lead to whole plant immunity via the mobilization of defense signals. One such signal is the plastid lipid-derived oxylipin azelaic acid (AZA). Arabidopsis lacking AZI1 or EARLI1, related lipid transfer family proteins, exhibit reduced AZA transport among leaves and cannot mount systemic immunity. AZA has been detected in roots as well as leaves. Therefore, the present study addresses the effects on plants of AZA application to roots. AZA but not the structurally related suberic acid inhibits root growth when directly in contact with roots. Treatment of roots with AZA also induces resistance to Pseudomonas syringae in aerial tissues. These effects of AZA on root growth and disease resistance depend, at least partially, on AZI1 and EARLI1. AZI1 in roots localizes to plastids, similar to its known location in leaves. Interestingly, kinases previously shown to modify AZI1 in vitro, MPK3 and MPK6, are also needed for AZA-induced root-growth inhibition and aboveground immunity. Finally, deuterium-labeled AZA applied to the roots does not move to aerial tissues. Thus, AZA application to roots triggers systemic immunity through an AZI1/EARLI1/MPK3/MPK6-dependent pathway and AZA effects may involve one or more additional mobile signals.

The nature of the progression of drought stress drives differential metabolomic responses in Populus deltoides.

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.

Genome-wide association studies and expression-based quantitative trait loci analyses reveal roles of HCT2 in caffeoylquinic acid biosynthesis and its regulation by defense-responsive transcription factors in Populus.

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.

Knockdown of a laccase in Populus deltoides confers altered cell wall chemistry and increased sugar release.

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

ALD1 Regulates Basal Immune Components and Early Inducible Defense Responses in Arabidopsis.

Robust immunity requires basal defense machinery to mediate timely responses and feedback cycles to amplify defenses against potentially spreading infections. AGD2-LIKE DEFENSE RESPONSE PROTEIN 1 (ALD1) is needed for the accumulation of the plant defense signal salicylic acid (SA) during the first hours after infection with the pathogen Pseudomonas syringae and is also upregulated by infection and SA. ALD1 is an aminotransferase with multiple substrates and products in vitro. Pipecolic acid (Pip) is an ALD1-dependent bioactive product induced by P. syringae. Here, we addressed roles of ALD1 in mediating defense amplification as well as the levels and responses of basal defense machinery. ALD1 needs immune components PAD4 and ICS1 (an SA synthesis enzyme) to confer disease resistance, possibly through a transcriptional amplification loop between them. Furthermore, ALD1 affects basal defense by controlling microbial-associated molecular pattern (MAMP) receptor levels and responsiveness. Vascular exudates from uninfected ALD1-overexpressing plants confer local immunity to the wild type and ald1 mutants yet are not enriched for Pip. We infer that, in addition to affecting Pip accumulation, ALD1 produces non-Pip metabolites that play roles in immunity. Thus, distinct metabolite signals controlled by the same enzyme affect basal and early defenses versus later defense responses, respectively.

Populus trichocarpa and Populus deltoides exhibit different metabolomic responses to colonization by the symbiotic fungus Laccaria bicolor.

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

Characterization of Clostridium thermocellum strains with disrupted fermentation end-product pathways.

Clostridium thermocellum is a thermophilic, cellulolytic anaerobe that is a candidate microorganism for industrial biofuels production. Strains with mutations in genes associated with production of L-lactate (Δldh) and/or acetate (Δpta) were characterized to gain insight into the intracellular processes that convert cellobiose to ethanol and other fermentation end-products. Cellobiose-grown cultures of the Δldh strain had identical biomass accumulation, fermentation end-products, transcription profile, and intracellular metabolite concentrations compared to its parent strain (DSM1313 Δhpt Δspo0A). The Δpta-deficient strain grew slower and had 30 % lower final biomass concentration compared to the parent strain, yet produced 75 % more ethanol. A Δldh Δpta double-mutant strain evolved for faster growth had a growth rate and ethanol yield comparable to the parent strain, whereas its biomass accumulation was comparable to Δpta. Free amino acids were secreted by all examined strains, with both Δpta strains secreting higher amounts of alanine, valine, isoleucine, proline, glutamine, and threonine. Valine concentration for Δldh Δpta reached 5 mM by the end of growth, or 2.7 % of the substrate carbon utilized. These secreted amino acid concentrations correlate with increased intracellular pyruvate concentrations, up to sixfold in the Δpta and 16-fold in the Δldh Δpta strain. We hypothesize that the deletions in fermentation end-product pathways result in an intracellular redox imbalance, which the organism attempts to relieve, in part by recycling NADP⁺ through increased production of amino acids.

Variable lignin structure revealed in Populus leaves.

Lignin has long been a trait of interest, especially in bioenergy feedstocks such as Populus. While the stem lignin of Populus is well studied, foliar lignin has received significantly less consideration. To this end, leaves from 11 field grown, natural variant Populus trichocarpa genotypes were investigated by NMR, FTIR, and GC-MS. Five of these genotypes were sufficiently irrigated, and the other six genotypes were irrigated at a reduced rate (59% of the potential evapotranspiration for the site) to induce drought treatment. Analysis by HSQC NMR revealed highly variable lignin structure among the samples, especially for the syringyl/guaiacyl (S/G) ratio, which ranged from 0.52-11.9. Appreciable levels of a condensed syringyl lignin structure were observed in most samples. The same genotype subjected to different treatments exhibited similar levels of condensed syringyl lignin, suggesting this was not a response to stress. A cross peak of δC/δH 74.6/5.03, consistent with the erythro form of the ß-O-4 linkage, was observed in genotypes where significant syringyl units were present. Principle component analysis revealed that FTIR absorbances associated with syringyl units (830 cm-1, 1317 cm-1) greatly contributed to variability between samples. Additionally, the ratio of 830/1230 cm-1 peak intensities were reasonably correlated (p-value < 0.05) with the S/G ratio determined by NMR. Analysis by GC-MS revealed significant variability of secondary metabolites such as tremuloidin, trichocarpin, and salicortin. Additionally, salicin derivatives were found to be well correlated with NMR results, which has been previously hypothesized. These results highlight previously unexplored nuance and variability associated with foliage tissue of poplar.