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Results for “Protein sequence analysis”

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At least 127 records · Page 7

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NASA NTRS1992

Development and use of domain-specific antibodies in a characterization of the large subunits of soybean photosystem 1

The molecular architecture of the soybean photosystem 1 reaction center complex was examined using a combination of surface labeling and immunological methodology on isolated thylakoid membranes. Synthetic peptides (12 to 14 amino acids in length) were prepared which correspond to the N-terminal regions of the 83 and 82.4 kDa subunits of photosystem 1 (the PsaA and PsaB proteins, respectively). Similarly, a synthetic peptide was prepared corresponding to the C-terminal region of the PsaB subunit. These peptides were conjugated to a carrier protein, and were used for the production of polyclonal antibodies in rabbits. The resulting sera could distinguish between the PsaA and PsaB photosystem 1 subunits by Western blot analysis, and could identify appropriate size classes of cyanogen bromide cleavage fragments as predicted from the primary sequences of these two subunits. When soybean thylakoid membranes were surface-labeled with N-hydroxysuccinimidobiotin, several subunits of the complete photosystem 1 lipid/protein complex incorporated label. These included the light harvesting chlorophyll proteins of photosystem 1, and peptides thought to aid in the docking of ferredoxin to the complex during photosynthetic electron transport. However, the PsaA and PsaB subunits showed very little biotinylation. When these subunits were examined for the domains to which biotin did attach, most of the observed label was associated with the N-terminal domain of the PsaA subunit, as identified using a domain-specific polyclonal antisera.

NASA Program NSCORT↗

DOE OSTI2024

A compact stem-loop DNA aptamer targets a uracil-binding pocket in the SARS-CoV-2 nucleocapsid RNA-binding domain

Abstract SARS-CoV-2 nucleocapsid (N) protein is a structural component of the virus with essential roles in the replication and packaging of the viral RNA genome. The N protein is also an important target of COVID-19 antigen tests and a promising vaccine candidate along with the spike protein. Here, we report a compact stem-loop DNA aptamer that binds tightly to the N-terminal RNA-binding domain of SARS-CoV-2 N protein. Crystallographic analysis shows that a hexanucleotide DNA motif (5′-TCGGAT-3′) of the aptamer fits into a positively charged concave surface of N-NTD and engages essential RNA-binding residues including Tyr109, which mediates a sequence-specific interaction in a uracil-binding pocket. Avid binding of the DNA aptamer allows isolation and sensitive detection of full-length N protein from crude cell lysates, demonstrating its selectivity and utility in biochemical applications. We further designed a chemically modified DNA aptamer and used it as a probe to examine the interaction of N-NTD with various RNA motifs, which revealed a strong preference for uridine-rich sequences. Our studies provide a high-affinity chemical probe for the SARS-CoV-2 N protein RNA-binding domain, which may be useful for diagnostic applications and investigating novel antiviral agents.

Biochemistry & Molecular Biology↗

DOE OSTI2021

Toho-1 β-lactamase: backbone chemical shift assignments and changes in dynamics upon binding with avibactam

Backbone chemical shift assignments for the Toho-1 β-lactamase (263 amino acids, 28.9 kDa) are reported based on triple resonance solution-state NMR experiments performed on a uniformly 2 H, 13 C, 15 N-labeled sample. These assignments allow for subsequent site-specific characterization at the chemical, structural, and dynamical levels. At the chemical level, titration with the non-β-lactam β-lactamase inhibitor avibactam is found to give chemical shift perturbations indicative of tight covalent binding that allow for mapping of the inhibitor binding site. At the structural level, protein secondary structure is predicted based on the backbone chemical shifts and protein residue sequence using TALOS-N and found to agree well with structural characterization from X-ray crystallography. At the dynamical level, model-free analysis of 15 N relaxation data at a single field of 16.4 T reveals well-ordered structures for the ligand-free and avibactam-bound enzymes with generalized order parameters of ~0.85. Complementary relaxation dispersion experiments indicate that there is an escalation in motions on the millisecond timescale in the vicinity of the active site upon substrate binding. The combination of high rigidity on short timescales and active site flexibility on longer timescales is consistent with hypotheses for achieving both high catalytic efficiency and broad substrate specificity: the induced active site dynamics allows variously sized substrates to be accommodated and increases the probability that the optimal conformation for catalysis will be sampled.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2025

Uncovering Sequence and Structural Characteristics of Fungal Expansin‐Related Proteins With Potential to Drive Substrate Targeting

Expansins loosen plant cell wall networks through disrupting non-covalent bonds between cellulose microfibrils and matrix polysaccharides. Whereas expansins were first discovered in plants, expansin-related proteins have since been identified in bacteria and fungi. The biological function of microbial expansins remains unclear; however, several studies have shown distinct binding preferences toward different structural polysaccharides. Earlier studies of bacterial expansin-related proteins uncovered sequence and structural features that correlate to substrate binding. Herein, 20 fungal expansin-related sequences were recombinantly produced in Komagataella phaffii, and the purified proteins were compared in terms of substrate binding to cellulosic and chitinous substrates. The impact of pH on the zeta potential of prioritized substrates was also measured, and Principal Component Analysis was performed to uncover correlations between protein characteristics (e.g., pI, hydrophobicity, surface charge distribution) and measured substrate binding preferences. Whereas acidic proteins with a predicted pI less than 5.0 preferentially bound to chitin, basic proteins with pI greater than 8.0 preferentially bound to xylan and xylan-containing fiber. Similar to many cellulases, binding to cellulose was correlated to relatively high aromatic amino acid content in the protein sequence and presence of a carbohydrate binding module (CBM), which in the case of expansins is a C-terminal CBM63. Whereas overall sequence characteristics could be correlated to substrate binding preference, the identity of amino acids occupying conserved positions that impact protein activity was better correlated with loosenin versus expansin classifications.

chitin↗

DOE OSTI2025

The promising role of proteomes and metabolomes in defining the single-cell landscapes of plants

The plant community has a strong track-record of RNA sequencing technology deployment, which combined with the recent advent of spatial platforms (e.g., 10x genomics), has resulted in an explosion of outstanding single cell and nuclei datasets that can be put in an in situ context within tissues (e.g., a cell atlas)1. In the genomics era, application of proteomics technologies in the plant sciences has always trailed behind that of RNA sequencing technologies, largely due to accessibility, ease-of-use and access to expertise along with depth of analysis benefits. On the other hand, the use of early analytical tools for characterizing small molecules (metabolites) from plant systems predates nucleic acid sequencing and proteomics analysis2, as the search for plant-based natural products has played a significant role in improving human health throughout history. However, the employment of proteomics and metabolomics assays for characterizing plant cell processes now remains significantly behind transcriptional approaches, even though both provide a direct functional readout of cell states and phenotypes.

Anderton, Christopher R. [BATTELLE (PACIFIC NW LAB↗

DOE OSTI2022

Top-down mass spectrometry and assigning internal fragments for determining disulfide bond positions in proteins

Disulfide bonds in proteins have a substantial impact on protein structure, stability, and biological activity. Localizing disulfide bonds is critical for understanding protein folding and higher-order structure. Conventional top-down mass spectrometry (TD-MS), where only terminal fragments are assigned for disulfide-intact proteins, can access disulfide information, but suffers from low fragmentation efficiency, thereby limiting sequence coverage. Here, we show that assigning internal fragments generated from TD-MS enhances the sequence coverage of disulfide-intact proteins by 20–60% by returning information from the interior of the protein sequence, which cannot be obtained by terminal fragments alone. Further, the inclusion of internal fragments can extend the sequence information of disulfide-intact proteins to near complete sequence coverage. Importantly, the enhanced sequence information that arise from the assignment of internal fragments can be used to determine the relative position of disulfide bonds and the exact disulfide connectivity between cysteines. The data presented here demonstrates the benefits of incorporating internal fragment analysis into the TD-MS workflow for analyzing disulfide-intact proteins, which would be valuable for characterizing biotherapeutic proteins such as monoclonal antibodies and antibody–drug conjugates.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

NASA NTRS2001

Atrogin-1, a muscle-specific F-box protein highly expressed during muscle atrophy

Muscle wasting is a debilitating consequence of fasting, inactivity, cancer, and other systemic diseases that results primarily from accelerated protein degradation by the ubiquitin-proteasome pathway. To identify key factors in this process, we have used cDNA microarrays to compare normal and atrophying muscles and found a unique gene fragment that is induced more than ninefold in muscles of fasted mice. We cloned this gene, which is expressed specifically in striated muscles. Because this mRNA also markedly increases in muscles atrophying because of diabetes, cancer, and renal failure, we named it atrogin-1. It contains a functional F-box domain that binds to Skp1 and thereby to Roc1 and Cul1, the other components of SCF-type Ub-protein ligases (E3s), as well as a nuclear localization sequence and PDZ-binding domain. On fasting, atrogin-1 mRNA levels increase specifically in skeletal muscle and before atrophy occurs. Atrogin-1 is one of the few examples of an F-box protein or Ub-protein ligase (E3) expressed in a tissue-specific manner and appears to be a critical component in the enhanced proteolysis leading to muscle atrophy in diverse diseases.

NASA Discipline Musculoskeletal↗

NASA NTRS1992

Antipeptide antibodies that can distinguish specific subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.)

The amino acid sequences of the beta and gamma subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.) root nodules are very similar. However, there are small regions within the sequences that are significantly different between the two polypeptides. The sequences between amino acids 2 and 9 and between 264 and 274 are examples. Three peptides (gamma 2-9, gamma 264-274, and beta 264-274) corresponding to these sequences were synthesized. Antibodies against these peptides were raised in rabbits and purified with corresponding peptide-Sepharose affinity chromatography. Western blot analysis of polyacrylamide gel electrophoresis of bean nodule proteins demonstrated that the anti-beta 264-274 antibodies reacted specifically with the beta polypeptide and the anti-gamma 264-274 and anti-gamma 2-9 antibodies reacted specifically with the gamma polypeptide of the native and denatured glutamine synthetase. These results showed the feasibility of using synthetic peptides in developing antibodies that are capable of distinguishing proteins with similar primary structures.

NASA Program NSCORT↗

DOE OSTI2023

Fecal microbiota transplantation ameliorates type 2 diabetes via metabolic remodeling of the gut microbiota in db/db mice

Gut microbiome (GM) deregulation has been implicated in major conditions such as obesity and type 2 diabetes (T2DM). Our previous prospective study indicated that fecal microbiota transplantation (FMT) successfully improved patients with T2DM. We hypothesized that FMT may be a potential therapeutic method for T2DM, but its precise mechanisms in T2DM remains to be elucidated. Eight db/m mice were FMT donors and control mice, and 16 genetically diabetic db/db mice were equally divided into two groups (db/db+phosphate-buffered saline (PBS) group, db/db+FMT group). The db/db+FMT group was administered fresh fecal suspension (0.2 mL/mice) daily for 4 weeks. Analysis of the GM and serum metabolome was carried out by 16S ribosomal RNA sequencing and liquid chromatogram-mass spectrometry, respectively. Effects of FMT on the gut barrier and pancreas were assessed using protein assays, messenger RNA, immunohistology and clinical indicators testing. Our results showed that FMT treatment of db/db mice relieves a series of clinical indicators, including fasting plasma glucose, serum insulin and oral glucose tolerance test among others. Compared with non-diabetic control mice, db/db+PBS mice exhibited decreased abundance of Ruminococaceae, Porphyromonadaceae and increased abundance of Rikenellaceae and Lactobacillaceae. FMT treatment reversed this effect on the microbiome. Eleven metabolites were changed between the db/db+PBS and db/db+FMT groups. Correlation analysis showed that the structural changes of the GM were correlated with host metabolite levels. We further showed that FMT treatment of db/db mice improved intestinal barrier function, reduced inflammation and caused an alteration in the number of circulating immune cells. FMT-mediated changes in the GM, serum metabolites, intestinal epithelial barrier, inflammation and circulating immune cells play an important role in the efficacy of FMT on T2DM disease progression.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2025

Design of diverse, functional mitochondrial targeting sequences across eukaryotic organisms using variational autoencoder

Mitochondria play a key role in energy production and metabolism, making them a promising target for metabolic engineering and disease treatment. However, despite the known influence of passenger proteins on localization efficiency, only a few protein-localization tags have been characterized for mitochondrial targeting. To address this limitation, we leverage a Variational Autoencoder to design novel mitochondrial targeting sequences. In silico analysis reveals that a high fraction of the generated peptides (90.14%) are functional and possess features important for mitochondrial targeting. We characterize artificial peptides in four eukaryotic organisms and, as a proof-of-concept, demonstrate their utility in increasing 3-hydroxypropionic acid titers through pathway compartmentalization and improving 5-aminolevulinate synthase delivery by 1.62-fold and 4.76-fold, respectively. Moreover, we employ latent space interpolation to shed light on the evolutionary origins of dual-targeting sequences. Overall, our work demonstrates the potential of generative artificial intelligence for both fundamental research and practical applications in mitochondrial biology.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Data for "Design of Diverse, Functional Mitochondrial Targeting Sequences Across Eukaryotic Organisms Using Variational Autoencoder"

AI/ML↗

NASA NTRS2001

The Role of Stress Proteins in Cell Stabilization: A Perspective from an Extremophile

The existence of organisms that live at near boiling temperatures is living proof that all of the complex biochemical machinery of life can be adapted to function under these harsh conditions. The purpose of our research is to elucidate the role of a group of proteins known as heat shock proteins or HSP60s in this adaptation to high temperatures. HSP60s are found in all organisms and they are among the most highly conserved proteins known. We are investigating HSP60s in an organism growing at 80 C and pH 2.0 (Sulfolobus shibatae). This organism produces three closely-related HSP60 proteins, referred to as HSP60 alpha, beta, and gamma. Our DOE-funded research during the last two years has focused on clarifying the role of FiSP60 alpha and beta. These are among the two most abundant proteins in S. shibatae grown at high temperatures and significantly increase in abundance when the cells are exposed to near-lethal temperatures. We have demonstrated that these proteins protect the cells from lethal temperatures by stabilizing their membranes. During this last year we have been studying gamma, which was discovered by genome sequence analysis but nothing was known about its function. We have determined that gamma is only expressed at low temperatures. that it interacts with alpha and beta, and that it influences their ability to form higher-order structures critical to their function. We propose that gamma modulates HSP60 function at low temperatures.

Trent, Jonathan↗

DOE OSTI2021

Evolutionary Diversification of Host-Targeted Bartonella Effectors Proteins Derived from a Conserved FicTA Toxin-Antitoxin Module

Proteins containing a FIC domain catalyze AMPylation and other post-translational modifications (PTMs). In bacteria, they are typically part of FicTA toxin-antitoxin modules that control conserved biochemical processes such as topoisomerase activity, but they have also repeatedly diversified into host-targeted virulence factors. Among these, Bartonella effector proteins (Beps) comprise a particularly diverse ensemble of FIC domains that subvert various host cellular functions. However, no comprehensive comparative analysis has been performed to infer molecular mechanisms underlying the biochemical and functional diversification of FIC domains in the vast Bep family. Here, we used X-ray crystallography, structural modelling, and phylogenetic analyses to unravel the expansion and diversification of Bep repertoires that evolved in parallel in three Bartonella lineages from a single ancestral FicTA toxin-antitoxin module. Our analysis is based on 99 non-redundant Bep sequences and nine crystal structures. Inferred from the conservation of the FIC signature motif that comprises the catalytic histidine and residues involved in substrate binding, about half of them represent AMP transferases. A quarter of Beps show a glutamate in a strategic position in the putative substrate binding pocket that would interfere with triphosphate-nucleotide binding but may allow binding of an AMPylated target for deAMPylation or another substrate to catalyze a distinct PTM. The β-hairpin flap that registers the modifiable target segment to the active site exhibits remarkable structural variability. The corresponding sequences form few well-defined groups that may recognize distinct target proteins. The binding of Beps to promiscuous FicA antitoxins is well conserved, indicating a role of the antitoxin to inhibit enzymatic activity or to serve as a chaperone for the FIC domain before translocation of the Bep into host cells. Taken together, our analysis indicates a remarkable functional plasticity of Beps that is mostly brought about by structural changes in the substrate pocket and the target dock. These findings may guide future structure–function analyses of the highly versatile FIC domains.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1998

Mouse osteoblastic cell line (MC3T3-E1) expresses extracellular calcium (Ca2+o)-sensing receptor and its agonists stimulate chemotaxis and proliferation of MC3T3-E1 cells

The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Osteoblasts appear at sites of osteoclastic bone resorption during bone remodeling in the "reversal" phase following osteoclastic resorption and preceding bone formation. Bone resorption produces substantial local increases in Ca2+o that could provide a signal for osteoblasts in the vicinity, leading us to determine whether such osteoblasts express the CaR. In this study, we used the mouse osteoblastic, clonal cell line MC3T3-E1. Both immunocytochemistry and Western blot analysis, using an antiserum specific for the CaR, detected CaR protein in MC3T3-E1 cells. We also identified CaR transcripts in MC3T3-E1 cells by Northern analysis using a CaR-specific riboprobe and by reverse transcription-polymerase chain reaction with CaR-specific primers, followed by nucleotide sequencing of the amplified products. Exposure of MC3T3-E1 cells to high Ca2+o (up to 4.8 mM) or the polycationic CaR agonists, neomycin and gadolinium (Gd3+), stimulated both chemotaxis and DNA synthesis in MC3T3-E1 cells. Therefore, taken together, our data strongly suggest that the osteoblastic cell line MC3T3-E1 possesses both CaR protein and mRNA very similar, if not identical, to those in parathyroid and kidney. Furthermore, the CaR in these osteoblasts could play a key role in regulating bone turnover by stimulating the proliferation and migration of such cells to sites of bone resorption as a result of local release of Ca2+o.

Non-NASA Center↗

DOE OSTI2024

Protein N -Glycans in Healthy and Sclerotic Glomeruli in Diabetic Kidney Disease

Diabetes is expected to directly affect renal glycosylation; yet to date, there has not been a comprehensive evaluation of alterations in N-glycan composition in the glomeruli of patients with diabetic kidney disease (DKD). Here, we used untargeted mass spectrometry imaging to identify N-glycan structures in healthy and sclerotic glomeruli in formalin-fixed paraffin-embedded sections from needle biopsies of five patients with DKD and three healthy kidney samples. Regional proteomics was performed on glomeruli from additional biopsies from the same patients to compare the abundances of enzymes involved in glycosylation. Secondary analysis of single-nucleus RNA sequencing (snRNAseq) data were used to inform on transcript levels of glycosylation machinery in different cell types and states. We detected 120 N-glycans, and among them, we identified 12 of these protein post-translated modifications that were significantly increased in glomeruli. All glomeruli-specific N-glycans contained an N-acetyllactosamine epitope. Five N-glycan structures were highly discriminant between sclerotic and healthy glomeruli. Sclerotic glomeruli had an additional set of glycans lacking fucose linked to their core, and they did not show tetra-antennary structures that were common in healthy glomeruli. Orthogonal omics analyses revealed lower protein abundance and lower gene expression involved in synthesizing fucosylated and branched N-glycans in sclerotic podocytes. In snRNAseq and regional proteomics analyses, we observed that genes and/or proteins involved in sialylation and N-acetyllactosamine synthesis were also downregulated in DKD glomeruli, but this alteration remained undetectable by our spatial N-glycomics assay. Integrative spatial glycomics, proteomics, and transcriptomics revealed protein N-glycosylation characteristic of sclerotic glomeruli in DKD.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2021

Developing Cost Effective Biological Removal Technology for Selenium and Nitrate from Flue Gas Desulfurization Wastewater from an Existing Power Facility

Funding from this project supported new research in environmental biotechnology at WVSU Energy and Environmental Science Institute (WVSUEESI) to develop biological treatment techniques for FGD wastewater using photosynthetic organisms algae and plants. We tested the possibility of using algae and plants in treating FGD wastewater. Results suggested that algae and plants could able to perform normal photosynthesis under low concentrations of selenium. However, higher concentrations of selenium are lethal on photosynthesis. The total protein data indicated that FGD wastewater induced the accumulation of complete proteins in the duckweeds compared to control plants. It appears that nitrate, nitrates, and other compounds in FGD wastewater promote total protein accumulation. The FGD wastewater treatment has triggered Triacylglycerols (TAG) and carbohydrate accumulation in duckweed. We studied the differential gene expression using RNA-Sequencing data. Gene expression analysis revealed that glycolysis, fatty acid, triacylglycerol biosynthesis, and starch degradation were up-regulated; on the contrary, gluconeogenesis, photosynthesis, and starch biosynthesis genes were down-regulated by FGD wastewater treatment. We confirmed the expression of some of these genes using q-RT PCR analysis. We demonstrated that when FGD gypsum was blended with the coal mine soil to grow the bioenergy crop, Camelina, it could grow and produce seed. Moreover, Camelina is a non-edible oilseed crop and has heavy metal sequestering properties, making it the right reclamation choice. Besides, this project provided the opportunity for WVSU undergraduates to integrate hands-on research and education. Funding from this grant helped us to enhance student hands-on experience in water quality and environmental science and participation in STEM research and education.

09 BIOMASS FUELS↗

DOE OSTI2020

Interlaboratory Study for Characterizing Monoclonal Antibodies by Top-Down and Middle-Down Mass Spectrometry

The Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications. To meet the needs of the rapidly growing therapeutic antibody market, it is important to develop analytical strategies to characterize the heterogeneity of a therapeutic product’s primary structure reliably and reproducibly. The major objective of the present study was to determine whether current TD/MD MS technologies and protocols can add value to the more commonly employed bottom-up approaches, such as confirming protein integrity, sequencing variable domains, ruling out artefacts, and revealing modifications and their locations. A panel of three mAbs was selected and centrally provided to twenty laboratories worldwide for the analysis: Sigma mAb standard (SiLuLite), NIST mAb standard, and the therapeutic mAb Herceptin (trastuzumab). A variety of different MS instrument platforms and ion dissociation techniques were employed. Overall, the present study confirms that MD/TD MS strategies are valuable and readily available tools in laboratories worldwide. They provide complementary information to the bottom-up approach and can add unique information, which may be crucial for comprehensive mAb characterization. The current limitations, as well as possible solutions to overcome them, are also outlined. One of the limitations revealed by the results of the present study is that the expert knowledge in both experiment and data analysis is indispensable to practice TD/MD MS, but the number of TD/MD MS experts is still very low.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Genome-wide identification and functional prediction of silicon (Si) transporters in poplar (Populus trichocarpa)

Abstract Silicon (Si) enhances plant tolerance to various biotic and abiotic stressors such as salinity, drought, and heat. In addition, Si can be biomineralized within plants to form organic carbon-containing phytoliths that can have ecosystem-level consequences by contributing to long-term carbon sequestration. Si is taken up and transported in plants via different transporter proteins such as influx transporters (e.g., Lsi1, Lsi6) and efflux transporters (e.g., Lsi2). Additionally, the imported Si can be deposited in plant leaves via silicification process using the Siliplant 1 (e.g., Slp1) protein. Functional homologs of these proteins have been reported in different food crops. Here, we performed a genome-wide analysis to identify different Si transporters and Slp1 homologs in the bioenergy crop poplar ( Populus trichocarpa Torr. and A. Gray ex W. Hook). We identified one channel-type Si influx transporter (PtLsi1; Potri.017G083300), one Si efflux transporter (PtLsi2; Potri.012G144000) and two proteins like Slp1 (PtSlp1a; Potri.004G168600 and PtSlp1b; Potri.009G129900 ) in the P. trichocarpa genome. We found a unique sequence (KPKPPVFKPPPVPI) in PtSlp1a which is repeated six times. Repeated presence of this sequence in PtSlp1a indicates that this protein might be important for silicification processes in P. trichocarpa. The mutation profiles of different Si transporters in a P. trichocarpa genome-wide association study population identified significant and impactful mutations in Potri.004G168600 and Potri.009G129900 . Using a publically accessible database ( http://bar.utoronto.ca/eplant_poplar/ ), digital expression analysis of the putative Si transporters in P. trichocarpa found low to moderate expression in the anticipated tissues, such as roots and leaves. Subcellular localization analysis found that PtLsi1/PtLsi2 are localized in the plasma membrane, whereas PtSlp1a/PtSlp1b are found in the extracellular spaces. Protein–Protein interaction analysis of PtLsi1/PtLsi2 identified Delta-1-pyrroline-5-carboxylate synthase (P5CS) as one of the main interacting partners of PtLsi2, which plays a key role in proline biosynthesis. Proline is a well-known participant in biotic and abiotic stress tolerance in plants. These findings will reinforce future efforts to modify Si accumulation for enhancing plant stress tolerance and carbon sequestration in poplar.

59 BASIC BIOLOGICAL SCIENCES↗