Expression Analysis of Auxin Regulated Genes in \u3cem\u3ePopulus\u3c/em\u3e

Due to its many advantageous characteristics, such as a small sequenced genome, ease of vegetative propagation and availability of genomic tools and databases, Populus is widely becoming accepted as the model species among trees. In addition, DOE has chosen hybrid poplar as the model bioenergy feedstock tree. Due to the growing importance of the Populus species, genetic and genomic resources (EST and BLAST databases, genetic maps, etc.) are becoming increasingly available and are leading to a greater understanding of the functionality of the Populus genome. The goal of this study was to use these resources to further characterize the genetic controls of root growth and development so that these mechanisms may eventually be manipulated to improve carbon sequestration ability in belowground sinks. Because auxin is known to play an important role in lateral root growth as well as many other aspects of plant development, a sequenced subtracted cDNA library from poplar was used to study the expression of genes up and down-regulated in response to exogenous auxin treatments. Results from this study indicate that a daily 9-day exogenous auxin treatment may have induced a stress response as indicated by the high percentage of WRKY transcription factors and stress related proteins that were up-regulated in response to the treatment regimen. A second study was also done using whole-genome oligonucleotide microarray technology to further analyze auxin regulated gene expression including Populus homologs of AUX/IAA and ARF genes in Arabidopsis. Results from this study did not appear to correlate well with real-time RT-PCR results indicating that, in the future, more reps need to be used to give the experiment the statistical power necessary to accurately find differentially expressed genes. Results from these gene expression studies can then be used to guide the development of poplar transgenics with increased root growth

Root Bacterial Endophytes Alter Plant Phenotype, but not Physiology

Plant traits, such as root and leaf area, influence how plants interact with their environment and the diverse microbiota living within plants can influence plant morphology and physiology. Here, we explored how three bacterial strains isolated from the Populus root microbiome, influenced plant phenotype. We chose three bacterial strains that differed in predicted metabolic capabilities, plant hormone production and metabolism, and secondary metabolite synthesis. We inoculated each bacterial strain on a single genotype of Populus trichocarpa and measured the response of plant growth related traits (root:shoot, biomass production, root and leaf growth rates) and physiological traits (chlorophyll content, net photosynthesis, net photosynthesis at saturating light–Asat, and saturating CO2–Amax). Overall, we found that bacterial root endophyte infection increased root growth rate up to 184% and leaf growth rate up to 137% relative to non-inoculated control plants, evidence that plants respond to bacteria by modifying morphology. However, endophyte inoculation had no influence on total plant biomass and photosynthetic traits (net photosynthesis, chlorophyll content). In sum, bacterial inoculation did not significantly increase plant carbon fixation and biomass, but their presence altered where and how carbon was being allocated in the plant host

Root bacterial endophytes alter plant phenotype, but not physiology

Plant traits, such as root and leaf area, influence how plants interact with their environment and the diverse microbiota living within plants can influence plant morphology and physiology. Here, we explored how three bacterial strains isolated from the Populus root microbiome, influenced plant phenotype. We chose three bacterial strains that differed in predicted metabolic capabilities, plant hormone production and metabolism, and secondary metabolite synthesis. We inoculated each bacterial strain on a single genotype of Populus trichocarpa and measured the response of plant growth related traits (root:shoot, biomass production, root and leaf growth rates) and physiological traits (chlorophyll content, net photosynthesis, net photosynthesis at saturating light–Asat, and saturating CO2–Amax). Overall, we found that bacterial root endophyte infection increased root growth rate up to 184% and leaf growth rate up to 137% relative to non-inoculated control plants, evidence that plants respond to bacteria by modifying morphology. However, endophyte inoculation had no influence on total plant biomass and photosynthetic traits (net photosynthesis, chlorophyll content). In sum, bacterial inoculation did not significantly increase plant carbon fixation and biomass, but their presence altered where and how carbon was being allocated in the plant host

Genome-wide analysis of lectin receptor-like kinases in Populus

Transcript level of C-type PtLecRLK gene in 24 different datasets from the Populus Gene Atlas Study. RNA-seq data were collected from the Populus Gene Atlas Study in Phytozome v11.0 ( http://phytozome.jgi.doe.gov/pz/portal.html ). The transcript level was expressed as FPKM. The sheet labeled as “whole_set” contains the original FPKM values from Gene Atlas. The data of four different tissues under standard condition are sorted in the data sheet labeled as “standard”. (XLSX 10 kb

Overexpression of a Prefoldin β 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

Nitrogen addition alters soil fungal communities, but root fungal communities are resistant to change

Plants are colonized by numerous microorganisms serving important symbiotic functions that are vital to plant growth and success. Understanding and harnessing these interactions will be useful in both managed and natural ecosystems faced with global change, but it is still unclear how variation in environmental conditions and soils influence the trajectory of these interactions. In this study, we examine how nitrogen addition alters plant-fungal interactions within two species of Populus - Populus deltoides and P. trichocarpa. In this experiment, we manipulated plant host, starting soil (native vs. away for each tree species), and nitrogen addition in a fully factorial replicated design. After ~10 weeks of growth, we destructively harvested the plants and characterized plant growth factors and the soil and root endosphere fungal communities using targeted amplicon sequencing of the ITS2 gene region. Overall, we found nitrogen addition altered plant growth factors, e.g., plant height, chlorophyll density, and plant N content. Interestingly, nitrogen addition resulted in a lower fungal alpha diversity in soils but not plant roots. Further, there was an interactive effect of tree species, soil origin, and nitrogen addition on soil fungal community composition. Starting soils collected from Oregon and West Virginia were dominated by the ectomycorrhizal fungi Inocybe (55.8% relative abundance), but interestingly when P. deltoides was grown in its native West Virginia soil, the roots selected for a high abundance of the arbuscular mycorrhizal fungi, Rhizophagus. These results highlight the importance of soil origin and plant species on establishing plant-fungal interactions

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

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

A New Calmodulin-Binding Protein Expresses in the Context of Secondary Cell Wall Biosynthesis and Impacts Biomass Properties in Populus

A greater understanding of biosynthesis, signaling and regulatory pathways involved in determining stem growth and secondary cell wall chemistry is important for enabling pathway engineering and genetic optimization of biomass properties. The present study describes a new functional role of PdIQD10, a Populus gene belonging to the IQ67-Domain1 family of IQD genes, in impacting biomass formation and chemistry. Expression studies showed that PdIQD10 has enhanced expression in developing xylem and tension-stressed tissues in Populus deltoides. Molecular dynamics simulation and yeast two-hybrid interaction experiments suggest interactions with two calmodulin proteins, CaM247 and CaM014, supporting the sequence-predicted functional role of the PdIQD10 as a calmodulin-binding protein. PdIQD10 was found to interact with specific Populus isoforms of the Kinesin Light Chain protein family, shown previously to function as microtubule-guided, cargo binding and delivery proteins in Arabidopsis. Subcellular localization studies showed that PdIQD10 localizes in the nucleus and plasma membrane regions. Promoter-binding assays suggest that a known master transcriptional regulator of secondary cell wall biosynthesis (PdWND1B) may be upstream of an HD-ZIP III gene that is in turn upstream of PdIQD10 gene in the transcriptional network. RNAi-mediated downregulation of PdIQD10 expression resulted in plants with altered biomass properties including higher cellulose, wall glucose content and greater biomass quantity. These results present evidence in support of a new functional role for an IQD gene family member, PdIQD10, in secondary cell wall biosynthesis and biomass formation in Populus

A carotenoid-deficient mutant in Pantoea sp. YR343, a bacteria isolated from the Rhizosphere of Populus deltoides, is defective in root colonization

The complex interactions between plants and their microbiome can have a profound effect on the health and productivity of the plant host. A better understanding of the microbial mechanisms that promote plant health and stress tolerance will enable strategies for improving the productivity of economically important plants. Pantoea sp. YR343 is a motile, rod-shaped bacterium isolated from the roots of Populus deltoides that possesses the ability to solubilize phosphate and produce the phytohormone indole-3-acetic acid (IAA). Pantoea sp. YR343 readily colonizes plant roots and does not appear to be pathogenic when applied to the leaves or roots of selected plant hosts. To better understand the molecular mechanisms involved in plant association and rhizosphere survival by Pantoea sp. YR343, we constructed a mutant in which the crtB gene encoding phytoene synthase was deleted. Phytoene synthase is responsible for converting geranylgeranyl pyrophosphate to phytoene, an important precursor to the production of carotenoids. As predicted, the ΔcrtB mutant is defective in carotenoid production, and shows increased sensitivity to oxidative stress. Moreover, we find that the ΔcrtB mutant is impaired in biofilm formation and production of IAA. Finally we demonstrate that the ΔcrtB mutant shows reduced colonization of plant roots. Taken together, these data suggest that carotenoids are important for plant association and/or rhizosphere survival in Pantoea sp. YR343.Work at the University of Notre Dame was supported by DOE grant SC0006642 (RM) and by a subcontract from Oak Ridge National Laboratory (SP).http://www.frontiersin.orgam2016Microbiology and Plant Patholog