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At least 721 records · Page 40
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Pith–specific lignification in Nicotiana attenuata as a defense against a stem–boring herbivore

Plants have developed tissue-specific defense strategies in response to various herbivores with different feeding habits. Although defense responses to leaf-chewing insects have been well studied, little is known about stem-specific responses, particularly in the pith, to stem-boring herbivores. To understand the stem-specific defense, we first conducted a comparative transcriptomic analysis of the wild tobacco Nicotiana attenuata before and after attack by the leaf-chewing herbivore Manduca sexta and the stem borer Trichobaris mucorea. When the stem-boring herbivore attacked, lignin-associated genes were upregulated specifically in the inner parenchymal cells of the stem, the pith; lignin also accumulated highly in the attacked pith. Silencing the lignin biosynthetic gene cinnamyl alcohol dehydrogenase enhanced the performance of the stem-boring herbivore but had no effect on the growth of the leaf-chewing herbivore. Two-dimensional nuclear magnetic resonance results revealed that lignified pith contains feruloyltyramine as an unusual lignin component in the cell wall, as a response against stem-boring herbivore attack. Pith-specific lignification induced by the stem-boring herbivore was modulated by both jasmonate and ethylene signaling. Furthermore, these results suggest that lignin provides a stem-specific inducible barrier, protecting plants against stem-boring insects.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Evolutionary innovations through gain and loss of genes in the ectomycorrhizal Boletales

Summary We aimed to identify genomic traits of transitions to ectomycorrhizal ecology within the Boletales by comparing the genomes of 21 symbiotrophic species with their saprotrophic brown‐rot relatives. Gene duplication rate is constant along the backbone of Boletales phylogeny with large loss events in several lineages, while gene family expansion sharply increased in the late Miocene, mostly in the Boletaceae. Ectomycorrhizal Boletales have a reduced set of plant cell‐wall‐degrading enzymes (PCWDEs) compared with their brown‐rot relatives. However, the various lineages retain distinct sets of PCWDEs, suggesting that, over their evolutionary history, symbiotic Boletales have become functionally diverse. A smaller PCWDE repertoire was found in Sclerodermatineae. The gene repertoire of several lignocellulose oxidoreductases (e.g. laccases) is similar in brown‐rot and ectomycorrhizal species, suggesting that symbiotic Boletales are capable of mild lignocellulose decomposition. Transposable element (TE) proliferation contributed to the higher evolutionary rate of genes encoding effector‐like small secreted proteins, proteases, and lipases. On the other hand, we showed that the loss of secreted CAZymes was not related to TE activity but to DNA decay. This study provides novel insights on our understanding of the mechanisms influencing the evolutionary diversification of symbiotic boletes.

Wu, Gang↗
DOE OSTI2022

A new approach to zip–lignin: 3,4–dihydroxybenzoate is compatible with lignification

Renewed interests in the development of bioenergy, biochemicals, and biomaterials have elicited new strategies for engineering the lignin of biomass feedstock plants. Furthermore, this study shows, for the first time, that 3,4-dihydroxybenzoate (DHB) is compatible with the radical coupling reactions that assemble polymeric lignin in plants. We introduced a bacterial 3-dehydroshikimate dehydratase into hybrid poplar (Populus alba × grandidentata) to divert carbon flux away from the shikimate pathway, which lies upstream of lignin biosynthesis. Transgenic poplar wood had up to 33% less lignin with p-hydroxyphenyl units comprising as much as 10% of the lignin. Mild alkaline hydrolysis of transgenic wood released fewer ester-linked p-hydroxybenzoate groups than control trees, and revealed the novel incorporation of cell-wall-bound DHB, as well as glycosides of 3,4-dihydroxybenzoic acid (DHBA). Two-dimensional nuclear magnetic resonance (2D-NMR) analysis uncovered DHBA-derived benzodioxane structures suggesting that DHB moieties were integrated into the lignin polymer backbone. In addition, up to 40% more glucose was released from transgenic wood following ionic liquid pretreatment and enzymatic hydrolysis. This work highlights the potential of diverting carbon flux from the shikimate pathway for lignin engineering and describes a new type of ‘zip-lignin’ derived from the incorporation of DHB into poplar lignin.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

BUZZ : an essential gene for postinitiation root hair growth and a mediator of root architecture in Brachypodium distachyon

Here, we discover a player in root development. Recovered from a forward-genetic screen in Brachypodium distachyon, the buzz mutant initiates root hairs but they fail to elongate. In addition, buzz roots grow twice as fast as wild-type roots. Also, lateral roots show increased sensitivity to nitrate, whereas primary roots are less sensitive to nitrate. Using whole-genome resequencing, we identified the causal single nucleotide polymorphism as occurring in a conserved but previously uncharacterized cyclin-dependent kinase (CDK)-like gene. The buzz mutant phenotypes are rescued by the wild-type B. distachyon BUZZ coding sequence and by an apparent homolog in Arabidopsis thaliana. Moreover, T-DNA mutants in A. thaliana BUZZ have shorter root hairs. BUZZ mRNA localizes to epidermal cells and develops root hairs and, in the latter, partially colocalizes with the NRT1.1A nitrate transporter. Based on qPCR and RNA-Seq, buzz overexpresses ROOT HAIRLESS LIKE SIX-1 and -2 and misregulates genes related to hormone signaling, RNA processing, cytoskeletal, and cell wall organization, and to the assimilation of nitrate. Overall, these data demonstrate that BUZZ is required for tip growth after root hair initiation and root architectural responses to nitrate.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Providing biological context for GWAS results using eQTL regulatory and co‐expression networks in Populus

Summary Our study utilized genome‐wide association studies (GWAS) to link nucleotide variants to traits in Populus trichocarpa , a species with rapid linkage disequilibrium decay. The aim was to overcome the challenge of interpreting statistical associations at individual loci without sufficient biological context, which often leads to reliance solely on gene annotations from unrelated model organisms. We employed an integrative approach that included GWAS targeting multiple traits using three individual techniques for lignocellulose phenotyping, expression quantitative trait loci (eQTL) analysis to construct transcriptional regulatory networks around each candidate locus and co‐expression analysis to provide biological context for these networks, using lignocellulose biosynthesis in Populus trichocarpa as a case study. The research identified three candidate genes potentially involved in lignocellulose formation, including one previously recognized gene (Potri.005G116800/VND1, a critical regulator of secondary cell wall formation) and two genes (Potri.012G130000/AtSAP9 and Potri.004G202900/BIC1) with newly identified putative roles in lignocellulose biosynthesis. Our integrative approach offers a framework for providing biological context to loci associated with trait variation, facilitating the discovery of new genes and regulatory networks.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Changes in leaf economic trait relationships across a precipitation gradient are related to differential gene expression in a C 4 perennial grass

Summary The leaf economics spectrum (LES) describes a suite of functional traits that consistently covary at large spatial and taxonomic scales. Despite its importance at these larger scales, few studies have examined the major drivers of intraspecific variation in the LES – phenotypic plasticity and standing genetic variation. Using experimental precipitation manipulations, we examined whether covariation among leaf economics traits and selection on leaf economics traits and trait combinations change as diverse genotypes of the widespread perennial grass Panicum virgatum are exposed to differences in precipitation. We also used RNA‐Seq to examine whether groups of co‐expressed genes that align with leaf economics traits function in processes hypothesized to underlie the LES. Water availability impacted leaf economics trait covariation in important ways – covariation between leaf economics traits and selection on covariation between traits (i.e. correlational selection) tended to be strongest when water availability was high. Additionally, many genes associated with leaf economics traits functioned in processes that may explain how the LES originates, such as chloroplasts, cell walls, and nitrogen metabolism. Water availability is likely an important modulator of selection and evolution of the LES in P. virgatum that can be better understood by examining gene expression.

Heckman, Robert W. [Department of Integrative Biol↗
DOE OSTI2020

The N–terminal zinc finger of CELLULOSE SYNTHASE6 is critical in defining its functional properties by determining the level of homodimerization in Arabidopsis

Primary cell wall cellulose is synthesized by the cellulose synthase complex (CSC) containing CELLULOSE SYNTHASE1 (CESA1), CESA3 and one of four CESA6–like proteins in Arabidopsis. It has been proposed that the CESA6–like proteins occupy the same position in the CSC, but their underlying selection mechanism remains unclear. We produced a chimeric CESA5 by replacing its N–terminal zinc finger with its CESA6 counterpart to investigate the consequences for its homodimerization, a crucial step in forming higher–order structures during assembly of the CSC. We found that the mutant phenotypes of prc1–1, a cesa6 null mutant, were rescued by the chimeric CESA5, and became comparable to the wild type (WT) and prc1–1 complemented by WT CESA6 in regard to plant growth, cellulose content, cellulose microfibril organization, CSC dynamics and subcellular localization. Bimolecular fluorescence complementation assays were employed to evaluate pairwise interactions between the N–terminal regions of CESA1, CESA3, CESA5, CESA6 and the chimeric CESA5. We verified that the chimeric CESA5 explicitly interacted with all the other CESA partners, comparable to CESA6, whereas interaction between CESA5 with itself was significantly weaker than that of all other CESA pairs. Furthermore, our findings suggest that the homodimerization of CESA6 through its N–terminal zinc finger is critical in defining its functional properties, and possibly determines its intrinsic roles in facilitating higher–order structures in CSCs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Rewired phenolic metabolism and improved saccharification efficiency of a Zea mays cinnamyl alcohol dehydrogenase 2 (zmcad2) mutant

Lignocellulosic biomass is an abundant byproduct from cereal crops that can potentially be valorized as a feedstock to produce biomaterials. Zea mays CINNAMYL ALCOHOL DEHYDROGENASE 2 (ZmCAD2) is involved in lignification, and is a promising target to improve the cellulose–to–glucose conversion of maize stover. Here, we analyzed a field–grown zmcad2 Mutator transposon insertional mutant. Zmcad2 mutant plants had an 18% lower Klason lignin content, whereas their cellulose content was similar to that of control lines. The lignin in zmcad2 mutants contained increased levels of hydroxycinnamaldehydes, i.e. the substrates of ZmCAD2, ferulic acid and tricin. Ferulates decorating hemicelluloses were not altered. Phenolic profiling further revealed that hydroxycinnamaldehydes are partly converted into (dihydro)ferulic acid and sinapic acid and their derivatives in zmcad2 mutants. Syringyl lactic acid hexoside, a metabolic sink in CAD–deficient dicot trees, appeared not to be a sink in zmcad2 maize. The enzymatic cellulose–to–glucose conversion efficiency was determined after 10 different thermochemical pre–treatments. Zmcad2 yielded significantly higher conversions compared with controls for almost every pre–treatment. However, the relative increase in glucose yields after alkaline pre–treatment was not higher than the relative increase when no pre–treatment was applied, suggesting that the positive effect of the incorporation of hydroxycinnamaldehydes was leveled off by the negative effect of reduced p–coumarate levels in the cell wall. Here, taken together, our results reveal how phenolic metabolism is affected in CAD–deficient maize, and further support mutating CAD genes in cereal crops as a promising strategy to improve lignocellulosic biomass for sugar–platform biorefineries.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Multiple Arabidopsis galacturonosyltransferases synthesize polymeric homogalacturonan by oligosaccharide acceptor‐dependent or de novo synthesis

SUMMARY Homogalacturonan (HG), the most abundant pectic glycan, functions as a cell wall structural and signaling molecule essential for plant growth, development and response to pathogens. HG exists as a component of pectic homoglycans, heteroglycans and glycoconjugates. HG is synthesized by members of the GALACTURONOSYLTRANSFERASE (GAUT) family. UDP‐GalA‐dependent homogalacturonan:galacturonosyltransferase (HG:GalAT) activity has previously been demonstrated for GAUTs 1, 4 and 11, as well as the GAUT1:GAUT7 complex. Here, we show that GAUTs 10, 13 and 14 are also HG:GalATs and that GAUTs 1, 10, 11, 13, 14 and 1:7 synthesize polymeric HG in vitro . Comparison of the in vitro HG:GalAT specific activities of the heterologously‐expressed proteins demonstrates GAUTs 10 and 11 with the lowest, GAUT1 and GAUT13 with moderate, and GAUT14 and the GAUT1:GAUT7 complex with the highest HG:GalAT activity. GAUT13 and GAUT14 are also shown to de novo synthesize (initiate) HG synthesis in the absence of exogenous HG acceptors, an activity previously demonstrated for GAUT1:GAUT7. The rate of de novo HG synthesis by GAUT13 and GAUT14 is similar to their acceptor dependent HG synthesis, in contrast to GAUT1:GAUT7 for which de novo synthesis occurred at much lower rates than acceptor‐dependent synthesis. The results suggest a unique role for de novo HG synthesis by GAUTs 13 and 14. The reducing end of GAUT13‐ de novo‐ synthesized HG has covalently attached UDP, indicating that UDP‐GalA serves as both a donor and acceptor substrate during de novo HG synthesis. The functional significance of unique GAUT HG:GalAT catalytic properties in the synthesis of different pectin glycan or glycoconjugate structures is discussed.

Engle, Kristen A.↗
DOE OSTI2022

Rerouting of the lignin biosynthetic pathway by inhibition of cytosolic shikimate recycling in transgenic hybrid aspen

Lignin is a phenolic polymer deposited in the plant cell wall and is mainly polymerized from three canonical monomers (monolignols), i.e., p-coumaryl, coniferyl, and sinapyl alcohols. After polymerization, these alcohols form different lignin substructures. In dicotyledons, monolignols are biosynthesized from phenylalanine, an aromatic amino acid. Shikimate acts at two positions in the route to the lignin building blocks. It is part of the shikimate pathway which provides the precursor for the biosynthesis of phenylalanine, and is involved in the transesterification of pcoumaroyl- CoA to p-coumaroyl shikimate, one of key steps in the biosynthesis of coniferyl and sinapyl alcohols. The shikimate residue in p-coumaroyl shikimate is released in later steps, and the resulting shikimate becomes available again for the biosynthesis of new p-coumaroyl shikimate molecules. In this study, we inhibited cytosolic shikimate recycling in transgenic hybrid aspen by accelerated phosphorylation of shikimate in the cytosol through expression of a bacterial shikimate kinase. Here, this expression elicited an increase in p-hydroxyphenyl units of lignin and, by contrast, a decrease in guaiacyl and syringyl units. Transgenic plants with high shikimate kinase activity produced a lignin content comparable to that in wild-type plants and had an increased processability via enzymatic saccharification. Although expression of many genes was altered in the transgenic plants, elevated shikimate kinase activity did not exert a significant effect on the expression of the majority of genes responsible for lignin biosynthesis. The present results indicate that cytosolic shikimate recycling is crucial to the monomeric composition of lignin rather than for lignin content.

shikimate pathway↗
DOE OSTI2024

Identification of glycosyltransferases mediating 2‐ O ‐arabinopyranosyl and 2‐ O ‐galactosyl substitutions of glucuronosyl side chains of xylan

SUMMARY Xylan is one of the major hemicelluloses in plant cell walls and its xylosyl backbone is often decorated at O ‐2 with glucuronic acid (GlcA) and/or methylglucuronic acid (MeGlcA) residues. The GlcA/MeGlcA side chains may be further substituted with 2‐ O ‐arabinopyranose (Ara p ) or 2‐ O ‐galactopyranose (Gal) residues in some plant species, but the enzymes responsible for these substitutions remain unknown. During our endeavor to investigate the enzymatic activities of Arabidopsis MUR3‐clade members of the GT47 glycosyltransferase family, we found that one of them was able to transfer Ara p from UDP‐Ara p onto O ‐2 of GlcA side chains of xylan, and thus it was named xylan 2‐ O ‐arabinopyranosyltransferase 1 (AtXAPT1). The function of AtXAPT1 was verified in planta by its T‐DNA knockout mutation showing a loss of the Ara p substitution on xylan GlcA side chains. Further biochemical characterization of XAPT close homologs from other plant species demonstrated that while the poplar ones had the same catalytic activity as AtXAPT1, those from Eucalyptus , lemon‐scented gum, sea apple, 'Ohi'a lehua, duckweed and purple yam were capable of catalyzing both 2‐ O ‐Ara p and 2‐ O ‐Gal substitutions of xylan GlcA side chains albeit with differential activities. Sequential reactions with XAPTs and glucuronoxylan methyltransferase 3 (GXM3) showed that XAPTs acted poorly on MeGlcA side chains, whereas GXM3 could efficiently methylate arabinosylated or galactosylated GlcA side chains of xylan. Furthermore, molecular docking and site‐directed mutagenesis analyses of Eucalyptus XAPT1 revealed critical roles of several amino acid residues at the putative active site in its activity. Together, these findings establish that XAPTs residing in the MUR3 clade of family GT47 are responsible for 2‐ O ‐arabinopyranosylation and 2‐ O ‐galactosylation of GlcA side chains of xylan.

Zhong, Ruiqin↗
DOE OSTI2025

Comparative transcriptomics uncovers poplar and fungal genetic determinants of ectomycorrhizal compatibility

Ectomycorrhizal symbiosis supports tree growth and is crucial for nutrient cycling and temperate and boreal ecosystems functioning. The establishment of functional ectomycorrhiza (ECM) first requires the association of compatible partners. However, host and fungal genetic determinants governing mycorrhizal compatibility are unknown. To identify such factors in poplar and its fungal associates, we mined existing and de novo tree and fungal transcriptional datasets. We identified co-expressed genes enabling ECM symbiosis at early and mature stages of the interaction. These sets of genes can be divided into general fungal-sensing and ECM-specific components. We highlight the importance of fungal modulation of plant JA-related defenses and the regulation of secretory pathways for ECM compatibility, including upregulation of key fungal small secreted proteins, the downregulation of plant secreted peroxidases, and the downregulation of plant cell wall remodeling proteins concomitantly with the upregulation of fungal glycosyl hydrolases acting on pectin. Not only gene regulation, but also its temporal scale and dynamics seem to play a crucial role for mycorrhizal compatibility. The expression profile of the host Common Symbiosis Pathway and nutrient transporters was also studied, revealing constitutive levels of expression and moderate upregulation in compatible ECM interactions. Overall, these results underscore the importance of novel biological functions during the establishment of ECM symbiosis, help us gain insights into the molecular events determining mycorrhiza compatibility, and serve as a data-rich transcriptomic resource to open new research questions in the field.

Marqués‐Gálvez, José Eduardo↗
DOE OSTI2024

Development of spectroscopic tools for nuclear forensics signatures development

The Savannah River National Laboratory (SRNL) has been developing advanced spectroscopic tools for the characterization of plutonium-bearing compounds with the intent to develop material and process signatures for nuclear forensics. Plutonium in a production, refining, or finishing facility will exist in many forms including oxide precursors (PuF4, PuF3, Pu oxalate, etc.), oxide, and metal. The ability to identify plutonium in each of these chemical forms and determine their processing history is crucial for the development of spectroscopic signatures. This presentation will focus on our work to develop and apply spectroscopic tools at SRNL using doubled-walled cells (DWC) to characterize the thermal decomposition of oxalates, calcination chemistry, alpha-decay-induced chemistry, age dating since last calcination, and other signatures related to plutonium processing.

Villa-Aleman, Eliel↗
DOE OSTI2022

Developmental changes in lignin composition are driven by both monolignol supply and laccase specificity

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

09 BIOMASS FUELS↗
DOE OSTI2022

Mechanism of mixed-linkage glucan biosynthesis by barley cellulose synthase–like CslF6 (1,3;1,4)-β-glucan synthase

Mixed-linkage (1,3;1,4)-β-glucans, which are widely distributed in cell walls of the grasses, are linear glucose polymers containing predominantly (1,4)-β-linked glucosyl units interspersed with single (1,3)-β-linked glucosyl units. Their distribution in cereal grains and unique structures are important determinants of dietary fibers that are beneficial to human health. We demonstrate that the barley cellulose synthase-like CslF6 enzyme is sufficient to synthesize a high–molecular weight (1,3;1,4)-β-glucan in vitro. Biochemical and cryo–electron microscopy analyses suggest that CslF6 functions as a monomer. A conserved “switch motif” at the entrance of the enzyme’s transmembrane channel is critical to generate (1,3)-linkages. There, a single-point mutation markedly reduces (1,3)-linkage formation, resulting in the synthesis of cellulosic polysaccharides. Our results suggest that CslF6 monitors the orientation of the nascent polysaccharide’s second or third glucosyl unit. Register-dependent interactions with these glucosyl residues reposition the polymer’s terminal glucosyl unit to form either a (1,3)- or (1,4)-β-linkage.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Molecular insights into the evolution of woody plant decay in the gut of termites

Plant cell walls represent the most abundant pool of organic carbon in terrestrial ecosystems but are highly recalcitrant to utilization by microbes and herbivores owing to the physical and chemical barrier provided by lignin biopolymers. Termites are a paradigmatic example of an organism’s having evolved the ability to substantially degrade lignified woody plants, yet atomic-scale characterization of lignin depolymerization by termites remains elusive. We report that the phylogenetically derived termite Nasutitermes sp. efficiently degrades lignin via substantial depletion of major interunit linkages and methoxyls by combining isotope-labeled feeding experiments and solution-state and solid-state nuclear magnetic resonance spectroscopy. Exploring the evolutionary origin of lignin depolymerization in termites, we reveal that the early-diverging woodroach Cryptocercus darwini has limited capability in degrading lignocellulose, leaving most polysaccharides intact. Conversely, the phylogenetically basal lineages of “lower” termites are able to disrupt the lignin-polysaccharide inter- and intramolecular bonding while leaving lignin largely intact. These findings advance knowledge on the elusive but efficient delignification in natural systems with implications for next-generation ligninolytic agents.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

A conserved glycan motif induces broadly reactive functional antibodies against the zoonotic pathogen Streptococcus suis

Streptococcus suis is a largely neglected but emerging bacterial zoonotic pathogen of global concern for animal welfare, antibiotic resistance development, and human health. No effective vaccines are now available. Here, we identified and characterized the function and structure of two cell wall polysaccharide variants in pathogenic S. suis strains using genetic deletion and (heterologous) complementation, lectin staining, glycan composition analysis, and specialized NMR spectroscopy. Both glycan variants were anionic polymers that differed in the presence of glucose in the side chain as a result of allelic variation in a glycosyltransferase gene. Deletion of this variable glycosyltransferase revealed an identical glycan “core” and affected S. suis morphology and lysozyme resistance. Immunization of pigs with this core domain elicited antibodies that recognized antigenically diverse pathogenic S. suis strains and induced complement deposition on encapsulated pathogenic S. suis strains. This study provides valuable insights for developing next-generation glycoconjugate vaccines, whereby a single-glycan target could protect against the emerging zoonotic pathogen S. suis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Loosenin-Like Proteins from Phanerochaete carnosa Impact Both Cellulose and Chitin Fiber Networks

The present study deepens investigations of microbial expansin-related proteins and their applied significance by (i) reporting a detailed comparison of diverse loosenins encoded by the same organism, (ii) considering both cellulosic and chitin-containing materials as targeted substrates, and (iii) investigating the impact of the C-terminal carbohydrate binding module (CBM) present in other expansin-related proteins on loosenin function. By revealing the potential of fungal loosenins to impact both cellulose and chitin-containing networks, our study reveals a possible biological and applied role of loosenins in fungal cell wall processing.

59 BASIC BIOLOGICAL SCIENCES↗