Molecular Remodeling in Populus PdKOR RNAi Roots Profiled Using LC-MS/MS Proteomics.

Plant endo-ß-1,4-glucanases belonging to the Glycoside Hydrolase Family 9 have functional roles in cell wall biosynthesis and remodeling via endohydrolysis of (1→4)-ß-d-glucosidic linkages. Modification of cell wall chemistry via RNA interference (RNAi)-mediated downregulation of Populus deltoides KORRIGAN (PdKOR), an endo-ß-1,4-glucanase familygene was shown to have functional consequences on the composition of secondary metabolome and the ability of modified roots to interact with beneficial microbes. The molecular remodeling that underlies the observed differences at metabolic, physiological, and morphological levels in roots is not well understood. Here a liquid chromatography (LC)-tandem mass spectrometry (MS/MS)-based proteome profiling approach is used to survey the molecular remodeling in root tissues of PdKOR and control plants. A total of 14316 peptides are identified and these mapped to 7139 P. deltoides proteins. Based on 90% sequence identity, the measured protein accessions represent 1187 functional protein groups. Analysis of Gene Ontology (GO) categories and specific individual proteins show differential expression of proteins relevant to plant-microbe interactions, cell wall chemistry, and metabolism. The new proteome dataset serves as a useful resource for deriving new hypotheses and empirical testing pertaining to functional roles of proteins and pathways in differential priming of plant roots to interactions with microbes.

Discovery and annotation of small proteins using genomics, proteomics, and computational approaches.

Small proteins (10-200 amino acids [aa] in length) encoded by short open reading frames (sORF) play important regulatory roles in various biological processes, including tumor progression, stress response, flowering, and hormone signaling. However, ab initio discovery of small proteins has been relatively overlooked. Recent advances in deep transcriptome sequencing make it possible to efficiently identify sORFs at the genome level. In this study, we obtained ~2.6 million expressed sequence tag (EST) reads from Populus deltoides leaf transcriptome and reconstructed full-length transcripts from the EST sequences. We identified an initial set of 12,852 sORFs encoding proteins of 10-200 aa in length. Three computational approaches were then used to enrich for bona fide protein-coding sORFs from the initial sORF set: (1) coding-potential prediction, (2) evolutionary conservation between P. deltoides and other plant species, and (3) gene family clustering within P. deltoides. As a result, a high-confidence sORF candidate set containing 1469 genes was obtained. Analysis of the protein domains, non-protein-coding RNA motifs, sequence length distribution, and protein mass spectrometry data supported this high-confidence sORF set. In the high-confidence sORF candidate set, known protein domains were identified in 1282 genes (higher-confidence sORF candidate set), out of which 611 genes, designated as highest-confidence candidate sORF set, were supported by proteomics data. Of the 611 highest-confidence candidate sORF genes, 56 were new to the current Populus genome annotation. This study not only demonstrates that there are potential sORF candidates to be annotated in sequenced genomes, but also presents an efficient strategy for discovery of sORFs in species with no genome annotation yet available.

Metabolic profiling reveals altered sugar and secondary metabolism in response to UGPase overexpression in Populus.

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

Systems and synthetic biology approaches to alter plant cell walls and reduce biomass recalcitrance.

Fine-tuning plant cell wall properties to render plant biomass more amenable to biofuel conversion is a colossal challenge. A deep knowledge of the biosynthesis and regulation of plant cell wall and a high-precision genome engineering toolset are the two essential pillars of efforts to alter plant cell walls and reduce biomass recalcitrance. The past decade has seen a meteoric rise in use of transcriptomics and high-resolution imaging methods resulting in fresh insights into composition, structure, formation and deconstruction of plant cell walls. Subsequent gene manipulation approaches, however, commonly include ubiquitous mis-expression of a single candidate gene in a host that carries an intact copy of the native gene. The challenges posed by pleiotropic and unintended changes resulting from such an approach are moving the field towards synthetic biology approaches. Synthetic biology builds on a systems biology knowledge base and leverages high-precision tools for high-throughput assembly of multigene constructs and pathways, precision genome editing and site-specific gene stacking, silencing and/or removal. Here, we summarize the recent breakthroughs in biosynthesis and remodelling of major secondary cell wall components, assess the impediments in obtaining a systems-level understanding and explore the potential opportunities in leveraging synthetic biology approaches to reduce biomass recalcitrance.

Involvement of auxin pathways in modulating root architecture during beneficial plant-microorganism interactions.

A wide variety of microorganisms known to produce auxin and auxin precursors form beneficial relationships with plants and alter host root development. Moreover, other signals produced by microorganisms affect auxin pathways in host plants. However, the precise role of auxin and auxin-signalling pathways in modulating plant-microbe interactions is unknown. Dissecting out the auxin synthesis, transport and signalling pathways resulting in the characteristic molecular, physiological and developmental response in plants will further illuminate upon how these intriguing inter-species interactions of environmental, ecological and economic significance occur. The present review seeks to survey and summarize the scattered evidence in support of known host root modifications brought about by beneficial microorganisms and implicate the role of auxin synthesis, transport and signal transduction in modulating beneficial effects in plants. Finally, through a synthesis of the current body of work, we present outstanding challenges and potential future research directions on studies related to auxin signalling in plant-microbe interactions.

Validation of a metabolite-GWAS network for Populus trichocarpa family 1 UDP-glycosyltransferases.

Metabolite genome-wide association studies (mGWASs) are increasingly used to discover the genetic basis of target phenotypes in plants such as Populus trichocarpa, a biofuel feedstock and model woody plant species. Despite their growing importance in plant genetics and metabolomics, few mGWASs are experimentally validated. Here, we present a functional genomics workflow for validating mGWAS-predicted enzyme-substrate relationships. We focus on uridine diphosphate-glycosyltransferases (UGTs), a large family of enzymes that catalyze sugar transfer to a variety of plant secondary metabolites involved in defense, signaling, and lignification. Glycosylation influences physiological roles, localization within cells and tissues, and metabolic fates of these metabolites. UGTs have substantially expanded in P. trichocarpa, presenting a challenge for large-scale characterization. Using a high-throughput assay, we produced substrate acceptance profiles for 40 previously uncharacterized candidate enzymes. Assays confirmed 10 of 13 leaf mGWAS associations, and a focused metabolite screen demonstrated varying levels of substrate specificity among UGTs. A substrate binding model case study of UGT-23 rationalized observed enzyme activities and mGWAS associations, including glycosylation of trichocarpinene to produce trichocarpin, a major higher-order salicylate in P. trichocarpa. We identified UGTs putatively involved in lignan, flavonoid, salicylate, and phytohormone metabolism, with potential implications for cell wall biosynthesis, nitrogen uptake, and biotic and abiotic stress response that determine sustainable biomass crop production. Our results provide new support for in silico analyses and evidence-based guidance for in vivo functional characterization.

Exploring the role of plant lysin motif receptor-like kinases in regulating plant-microbe interactions in the bioenergy crop Populus.

For plants, distinguishing between mutualistic and pathogenic microbes is a matter of survival. All microbes contain microbe-associated molecular patterns (MAMPs) that are perceived by plant pattern recognition receptors (PRRs). Lysin motif receptor-like kinases (LysM-RLKs) are PRRs attuned for binding and triggering a response to specific MAMPs, including chitin oligomers (COs) in fungi, lipo-chitooligosaccharides (LCOs), which are produced by mycorrhizal fungi and nitrogen-fixing rhizobial bacteria, and peptidoglycan in bacteria. The identification and characterization of LysM-RLKs in candidate bioenergy crops including Populus are limited compared to other model plant species, thus inhibiting our ability to both understand and engineer microbe-mediated gains in plant productivity. As such, we performed a sequence analysis of LysM-RLKs in the Populus genome and predicted their function based on phylogenetic analysis with known LysM-RLKs. Then, using predictive models, molecular dynamics simulations, and comparative structural analysis with previously characterized CO and LCO plant receptors, we identified probable ligand-binding sites in Populus LysM-RLKs. Using several machine learning models, we predicted remarkably consistent binding affinity rankings of Populus proteins to CO. In addition, we used a modified Random Walk with Restart network-topology based approach to identify a subset of Populus LysM-RLKs that are functionally related and propose a corresponding signal transduction cascade. Our findings provide the first look into the role of LysM-RLKs in Populus-microbe interactions and establish a crucial jumping-off point for future research efforts to understand specificity and redundancy in microbial perception mechanisms.

Biomass formation and sugar release efficiency of Populus modified by altered expression of a NAC transcription factor.

Woody biomass is an important feedstock for biofuel production. Manipulation of wood properties that enable efficient conversion of biomass to biofuel reduces cost of biofuel production. Wood cell wall composition is regulated at several levels that involve expression of transcription factors such as wood-/secondary cell wall-associated NAC domains (WND or SND). In Arabidopsis thaliana, SND1 regulates cell wall composition through activation of its down-stream targets such as MYBs. The functional aspects of SND1 homologs in the woody Populus have been studied through transgenic manipulation. In this study, we investigated the role of PdWND1B, Populus SND1 sequence ortholog, in wood formation using transgenic manipulation through over-expression or silencing under the control of a vascular-specific 4-coumarate-CoA ligase (4CL) promoter. As compared with control plants, PdWND1B-RNAi plants were shorter in height, with significantly reduced stem diameter and dry biomass, whereas there were no significant differences in growth and productivity of PdWND1B over-expression plants. Conversely, PdWND1B over-expression lines showed a significant reduction in cellulose and increase in lignin content, whereas there was no significant impact on lignin content of downregulated lines. Stem carbohydrate composition analysis revealed a decrease in glucose, mannose, arabinose, and galactose, but an increase in xylose in the over-expression lines. Transcriptome analysis revealed upregulation of several downstream transcription factors and secondary cell wall related structural genes in the PdWND1B over-expression lines, partly explaining the observed phenotypic changes in cell wall chemistry. Relative to the control, glucose release efficiency and ethanol production from stem biomass was significantly reduced in over-expression lines. Our results show that PdWND1B is an important factor determining biomass productivity, cell wall chemistry and its conversion to biofuels in Populus.

Challenges of the utilization of wood polymers: how can they be overcome?

Diminishing fossil fuel resources as well as growing environmental and energy security concerns, in parallel with growing demands on raw materials and energy, have intensified global efforts to utilize wood biopolymers as a renewable resource to produce biofuels and biomaterials. Wood is one of the most abundant biopolymer composites on earth that can be converted into biofuels as well as used as a platform to produce bio-based materials. The major biopolymers in wood are cellulose, hemicelluloses, and lignin which account for >90% of dry weight. These polymers are generally associated with each other in wood cell walls resulting in an intricate and dynamic cell wall structure. This mini-review provides an overview of major wood biopolymers, their structure, and recent developments in their utilization to develop biofuels. Advances in genetic modifications to overcome the recalcitrance of woody biomass for biofuels are discussed and point to a promising future.

Importance of suberin biopolymer in plant function, contributions to soil organic carbon and in the production of bio-derived energy and materials.

Suberin is a hydrophobic biopolymer of significance in the production of biomass-derived materials and in biogeochemical cycling in terrestrial ecosystems. Here, we describe suberin structure and biosynthesis, and its importance in biological (i.e., plant bark and roots), ecological (soil organic carbon) and economic (biomass conversion to bioproducts) contexts. Furthermore, we highlight the genomics and analytical approaches currently available and explore opportunities for future technologies to study suberin in quantitative and/or high-throughput platforms in bioenergy crops. A greater understanding of suberin structure and production in lignocellulosic biomass can be leveraged to improve representation in life cycle analysis and techno-economic analysis models and enable performance improvements in plant biosystems as well as informed crop system management to achieve economic and environmental co-benefits.

Biological Parts for Plant Biodesign to Enhance Land-Based Carbon Dioxide Removal.

A grand challenge facing society is climate change caused mainly by rising CO2 concentration in Earth's atmosphere. Terrestrial plants are linchpins in global carbon cycling, with a unique capability of capturing CO2 via photosynthesis and translocating captured carbon to stems, roots, and soils for long-term storage. However, many researchers postulate that existing land plants cannot meet the ambitious requirement for CO2 removal to mitigate climate change in the future due to low photosynthetic efficiency, limited carbon allocation for long-term storage, and low suitability for the bioeconomy. To address these limitations, there is an urgent need for genetic improvement of existing plants or construction of novel plant systems through biosystems design (or biodesign). Here, we summarize validated biological parts (e.g., protein-encoding genes and noncoding RNAs) for biological engineering of carbon dioxide removal (CDR) traits in terrestrial plants to accelerate land-based decarbonization in bioenergy plantations and agricultural settings and promote a vibrant bioeconomy. Specifically, we first summarize the framework of plant-based CDR (e.g., CO2 capture, translocation, storage, and conversion to value-added products). Then, we highlight some representative biological parts, with experimental evidence, in this framework. Finally, we discuss challenges and strategies for the identification and curation of biological parts for CDR engineering in plants.

Cultivating the Bacterial Microbiota of Populus Roots.

The integral role of microbial communities in plant growth and health is now widely recognized, and, increasingly, the constituents of the microbiome are being defined. While phylogenetic surveys have revealed the taxa present in a microbiome and show that this composition can depend on, and respond to, environmental perturbations, the challenge shifts to determining why particular microbes are selected and how they collectively function in concert with their host. In this study, we targeted the isolation of representative bacterial strains from environmental samples of Populus roots using a direct plating approach and compared them to amplicon-based sequencing analysis of root samples. The resulting culture collection contains 3,211 unique isolates representing 10 classes, 18 orders, 45 families, and 120 genera from 6 phyla, based on 16S rRNA gene sequence analysis. The collection accounts for ∼50% of the natural community of plant-associated bacteria as determined by phylogenetic analysis. Additionally, a representative set of 553 had their genomes sequenced to facilitate functional analyses. The top sequence variants in the amplicon data, identified as Pseudomonas, had multiple representatives within the culture collection. We then explore a simplified microbiome, comprised of 10 strains representing abundant taxa from environmental samples, and tested for their ability to reproducibly colonize Populus root tissue. The 10-member simplified community was able to reproducibly colonize on Populus roots after 21 days, with some taxa found in surface-sterilized aboveground tissue. This study presents a comprehensive collection of bacteria isolated from Populus for use in exploring microbial function and community inoculation experiments to understand basic concepts of plant and environmental selection. IMPORTANCE Microbial communities play an integral role in the health and survival of their plant hosts. Many studies have identified key members in these communities and led to the use of synthetic communities for elucidating their function; however, these studies are limited by the available cultured bacterial representatives. Here, we present a bacterial culture collection comprising 3,211 isolates that is representative of the root community of Populus. We then demonstrate the ability to examine underlying microbe-microbe interactions using a synthetic community approach. This culture collection will allow for the greater exploration of the microbial community function through targeted experimentation and manipulation.

Plant Biosystems Design for a Carbon-Neutral Bioeconomy.

Our society faces multiple daunting challenges including finding sustainable solutions towards climate change mitigation; efficient production of food, biofuels, and biomaterials; maximizing land-use efficiency; and enabling a sustainable bioeconomy. Plants can provide environmentally and economically sustainable solutions to these challenges due to their inherent capabilities for photosynthetic capture of atmospheric CO2, allocation of carbon to various organs and partitioning into various chemical forms, including contributions to total soil carbon. In order to enhance crop productivity and optimize chemistry simultaneously in the above- and belowground plant tissues, transformative biosystems design strategies are needed. Concerted research efforts will be required for accelerating the development of plant cultivars, genotypes, or varieties that are cooptimized in the contexts of biomass-derived fuels and/or materials aboveground and enhanced carbon sequestration belowground. Here, we briefly discuss significant knowledge gaps in our process understanding and the potential of synthetic biology in enabling advancements along the fundamental to applied research arc. Ultimately, a convergence of perspectives from academic, industrial, government, and consumer sectors will be needed to realize the potential merits of plant biosystems design for a carbon neutral bioeconomy.

Draft Genome Sequence of Larkinella sp. Strain BK230, Isolated from Populus deltoides Roots.

Larkinella sp. strain BK230, a heterotrophic bacterium of the phylum Bacteroidetes, was isolated from the roots of a field-grown eastern cottonwood tree (Populus deltoides) located in Georgia. The draft 7.27-Mb genome has a G+C content of 53.4% and contains 6,026 coding sequences, including 41 tRNA genes.

Draft Genome Sequence of Tumebacillus sp. Strain BK434, Isolated from the Roots of Eastern Cottonwood.

A Gram-positive bacterium was isolated from the root of an eastern cottonwood tree (Populus deltoides) in Georgia and identified as a Tumebacillus species with 99% 16S rRNA nucleotide identity to Tumebacillus avium The genome is 4.6 Mbp and encodes 4,072 proteins and 251 RNAs.

Nanomechanics and Raman Spectroscopy of in Situ Native Carbohydrate Storage Granules for Enhancing Starch Quality and Lignocellulosic Biomass Production.

Alternative energy strategies based on plant biomass-derived bioenergy and biofuels rely on understanding and optimization of plant structure, chemistry, and performance. Starch, a constitutive element of all green plants, is important to food, biofuels, and industrial applications. Models of carbohydrate storage granules are highly heterogeneous in representing morphology and structure, though a deeper understanding of the role of structure in functional behavior is emerging. A better understanding of the in situ nanoscale properties of native granules is needed to help improve the starch quality in food crops as well as optimize lignocellulosic biomass production in perennial nonfood crops. Here, we present a new technique called soft mechanical nano-ablation (sMNA) for accessing the interior of the granules without compromising the inner nanostructure. We then explore the nanomechanics of granules within the ray parenchyma cells of Populus xylem, a desirable woody biofuel feedstock. The employed soft outer layer nanoablation and atomic force microscopy reveal that the inner structure comprises 156 nm blocklets arranged in a semicrystalline organization. The nanomechanical properties of the inner and outer structures of a single starch granule are measured and found to exhibit large variations, changing by a factor of 3 in Young's modulus and a factor of 2 in viscoplastic index. These findings demonstrate how the introduced approach facilitates studies of structure-function relationships among starch granules and more complex secondary cell wall features as they relate to plant performance.

Transgenic Poplar Designed for Biofuels.

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

Plant Biosystems Design Research Roadmap 1.0.

Human life intimately depends on plants for food, biomaterials, health, energy, and a sustainable environment. Various plants have been genetically improved mostly through breeding, along with limited modification via genetic engineering, yet they are still not able to meet the ever-increasing needs, in terms of both quantity and quality, resulting from the rapid increase in world population and expected standards of living. A step change that may address these challenges would be to expand the potential of plants using biosystems design approaches. This represents a shift in plant science research from relatively simple trial-and-error approaches to innovative strategies based on predictive models of biological systems. Plant biosystems design seeks to accelerate plant genetic improvement using genome editing and genetic circuit engineering or create novel plant systems through de novo synthesis of plant genomes. From this perspective, we present a comprehensive roadmap of plant biosystems design covering theories, principles, and technical methods, along with potential applications in basic and applied plant biology research. We highlight current challenges, future opportunities, and research priorities, along with a framework for international collaboration, towards rapid advancement of this emerging interdisciplinary area of research. Finally, we discuss the importance of social responsibility in utilizing plant biosystems design and suggest strategies for improving public perception, trust, and acceptance.

Shotgun proteome profile of Populus developing xylem.

Understanding the molecular pathways of plant cell wall biosynthesis and remodeling is central to interpreting biological mechanisms underlying plant growth and adaptation as well as leveraging that knowledge towards development of improved bioenergy feedstocks. Here, we report the application of shotgun MS/MS profiling to the proteome of Populus developing xylem. Nearly 6000 different proteins were identified from the xylem proteome. To identify low-abundance DNA-regulatory proteins from the developing xylem, a selective nuclear proteome profiling method was developed. Several putative transcription factors and chromatin remodeling proteins were identified using this method, such as NAC domain, CtCP-like and CHB3-SWI/SNF-related proteins. Public databases were mined to obtain information in support of subcellular localization, transcript-level expression and functional categorization of identified proteins. In addition to finding protein-level evidence of candidate cell wall biosynthesis genes from xylem (wood) tissue such as cellulose synthase, sucrose synthase and polygalacturonase, several other potentially new candidate genes in the cell wall biosynthesis pathway were discovered. Further application of such proteomics methods will aid in plant systems biology modeling efforts by enhancing the understanding not only of cell wall biosynthesis but also of other plant developmental and physiological pathways.

Tension wood structure and morphology conducive for better enzymatic digestion.

BACKGROUND: Tension wood is a type of reaction wood in response to bending or leaning stem as a corrective growth process. Tension wood is formed by both natural and man-made processes. Most attractively, tension wood contains higher glucan content and undergoes higher enzymatic conversion to fermentable sugars. Here, we have employed structural techniques, small-angle neutron scattering (SANS) and wide-angle X-ray diffraction (WAXD) to elucidate structural and morphological aspects of tension wood conducive to higher sugar yields. RESULTS: Small-angle neutron scattering data exhibited a tri-modal distribution of the fibril cross-sectional dimension. The smallest size, 22 Å observed in all samples concurred with the WAXD results of the control and opposite side samples. This smallest and the most abundant occurring size was interpreted as the cellulose elementary microfibril diameter. The intermediate size of 45 Å, which is most pronounced in the tension side sample and consistent with WAXD results for tension side sample, indicates association of neighboring elementary microfibrils to form larger crystallite bundles. The largest size 61 Å observed by SANS was however not observed by WAXD and therefore associated to mesopores. CONCLUSIONS: Structure and morphology of tension wood is different from control wood. Cellulose crystallinity increases, lignin content is lower and the appearance of mesopores with 61 Å diameter is observed. Despite the presence of higher crystalline cellulose content in tension side, the lower lignin content and may be combined with the abundance of mesopores, substantially improves enzyme accessibility leading to higher yields in cellulose digestion.