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All sources NASA NTRS DOE OSTI

DOE OSTI2018

Substrate-Specific Differential Gene Expression and RNA Editing in the Brown Rot Fungus Fomitopsis pinicola

Wood-decaying fungi tend to have characteristic substrate ranges that partly define their ecological niche. Fomitopsis pinicola is a brown rot species of Polyporales that is reported on 82 species of softwoods and 42 species of hardwoods. We analyzed the gene expression levels and RNA editing profiles of F. pinicola from submerged cultures with ground wood powder (sampled at 5 days) or solid wood wafers (sampled at 10 and 30 days), using aspen, pine, and spruce substrates (aspen was used only in submerged cultures). Fomitopsis pinicola expressed similar sets of wood-degrading enzymes typical of brown rot fungi across all culture conditions and time points. Nevertheless, differential gene expression and RNA editing were observed across all pairwise comparisons of substrates and time points. Genes exhibiting differential expression and RNA editing encode diverse enzymes with known or potential function in brown rot decay, including laccase, benzoquinone reductase, aryl alcohol oxidase, cytochrome P450s, and various glycoside hydrolases. There was no overlap between differentially expressed and differentially edited genes, suggesting that these may provide F. pinicola with independent mechanisms for responding to different conditions. Comparing transcriptomes from submerged cultures and wood wafers, we found that culture conditions had a greater impact on global expression profiles than substrate wood species. In contrast, the suites of genes subject to RNA editing were much less affected by culture conditions. These findings highlight the need for standardization of culture conditions in studies of gene expression in wood-decaying fungi.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2018

Physiological and genomic signatures of evolutionary thermal adaptation in redband trout from extreme climates

Abstract Temperature is a master environmental factor that limits the geographical distribution of species, especially in ectotherms. To address challenges in biodiversity conservation under ongoing climate change, it is essential to characterize relevant functional limitations and adaptive genomic content at population and species levels. Here, we present evidence for adaptive divergence in cardiac function and genomic regions in redband trout ( Oncorhynchus mykiss gairdneri ) populations from desert and montane streams. Cardiac phenotypes of individual fish were measured in the field with a custom‐built electrocardiogram apparatus. Maximum heart rate and its rate limiting temperature during acute warming were significantly higher in fish that have evolved in the extreme of a desert climate compared to a montane climate. Association mapping with 526,301 single nucleotide polymorphisms ( SNP s) across the genome revealed signatures of thermal selection both within and among ecotypes. Among desert and montane populations, 435 SNP s were identified as putative outliers under natural selection and 20 of these loci showed significant association with average summer water temperatures among populations. Phenotypes for cardiac performance were variable within each ecotype, and 207 genomic regions were strongly associated with either maximum heart rate or rate limiting temperatures among individuals. Annotation of significant loci provided candidate genes that underlie thermal adaptation, including pathways associated with cardiac function ( IRX 5, CASQ 1, CAC 1D, and TITIN ), neuroendocrine system ( GPR 17 and NOS ), and stress response ( SERPH ). By integrating comparative physiology and population genomics, results here advance our knowledge on evolutionary processes of thermal adaptation in aquatic ectotherms.

Chen, Zhongqi↗

NASA NTRSDate not supplied

INFLUENCE OF ACTN3 GENE ON MUSCLE HEALTH AND PHYSICAL FITNESS TO INFORM FUTURE INTERVENTIONS IN SPACEFLIGHT MISSIONS

INTRODUCTION: Exposure to reduced gravity environments leads to diminished muscle size, strength, and endurance. Preservation of physical fitness is critical for mission essential tasks such as extravehicular activities or adaptation to changes in gravity loads. Current countermeasures to maintain ISS astronauts’ muscle mass and fitness include dedicated time for a combination of cardiovascular exercise and resistance training. Countermeasure effectiveness is monitored through VO2 max and isometric mid-thigh pulls. It is important to understand both environmental and genetic contributors to astronaut physical fitness. Multiple genes, including ACTN3, are known to correlate with exercise phenotypes and have the potential to help personalize countermeasures based on an astronaut’s genotypic makeup. TOPIC: The ACTN3 gene encodes a protein expressed only in fast-twitch muscles and correlates with sprint and power phenotypes. A common polymorphism in ACTN3 gene is R577X (rs1815739), produced by a C−to−T base substitution resulting in a nonsense mutation from arginine (R) to a premature stop codon (X) present in approximately 18% of the population. This polymorphism causes an absence of α−actinin−3 in type II muscle fibers but does not lead to a disease phenotype. The RR genotype is associated with elite athletes, especially in sprint and power sports. The XX genotype is believed to be more common in endurance athletes. The ACTN3 gene also has potential associations with training adaptation, post-exercise recovery, and exercise-associated injuries. APPLICATION: Understanding how ACTN3 and other genetic determinates of fitness phenotypes affect astronaut physical performance can inform personalized exercise and recovery prescriptions. For example, individuals with the RR or RX genotype may respond better to high-load and low repetition exercise, while XX may benefit from high repetition with low weight exercise. Since the wild-type protein may confer more resistance to muscle damage, those individuals with the RR or RX genotype may benefit from high intensity interval training for improved VO2 max, whereas those with an XX genotype may benefit from low intensity, high volume endurance activity. Additionally, omics data related to health and performance could be used as biomarkers to monitor astronaut fitness and the effectiveness of training regimens during a mission. Learning objectives: • Learn about the phenotypic effects on muscle function and fitness secondary to R577X mutation in the ACTN3 gene • Learn how mutations in the ACTN3 gene may inform future countermeasures for future spaceflight missions

Lynn K Stanwyck↗

DOE OSTI2021

Endothelial cells control vascular smooth muscle cell cholesterol levels by regulating 24-dehydrocholesterol reductase expression

Highlights: • Suspended endothelial and vascular smooth muscle cells organize to form spherical vascular organoids in hanging drops. • Vascular smooth muscle cells downregulate the expression of metabolism regulating genes in presence of endothelial cells. • Endothelial cells decrease the levels of 24-dehydrocholesterol reductase and cholesterol in vascular smooth muscle cells. • Diminished cholesterol biosynthesis inhibits vascular smooth muscle cell activity. Communication of vascular cells is essential for the control of organotypic functions of blood vessels. In this context, vascular endothelial cells (EC) act as potent regulators of vascular smooth muscle cell (VSMC) functions such as contraction and relaxation. However, the impact of ECs on the gene expression pattern of VSMCs is largely unknown. Here, we investigated changes of the VSMC transcriptome by utilizing 3D human vascular organoids organized as a core of VSMCs enclosed by a monolayer of ECs. Microarray-based analyses indicated that interaction with ECs for 48 h down-regulates expression of genes in VSMCs controlling rate-limiting steps of the cholesterol biosynthesis such as HMGCR, HMGCS1, DHCR24 and DHCR7. Protein analyses revealed a decrease in the abundance of DHCR24 (24-dehydrocholesterol reductase) and lower cholesterol levels in VSMCs co-cultured with ECs. On the functional level, the blockade of the DHCR24 activity impaired adhesion, migration and proliferation of VSMCs. Collectively, these findings indicate that ECs have the capacity to instruct VSMCs to shut down the expression of DHCR24 thereby limiting their cholesterol biosynthesis, which may support their functional steady state.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2019

GENE-43. Targeting GABPb1L Inhibits In Vivo Growth of TERT Promoter Mutant Glioblastoma

Understanding cancer cell immortality in primary glioblastoma (GBM) is essential for the development of more informed treatments. Multiple cancer types, including >80% of GBMs, undergo immortalization by reactivating Telomerase Reverse Transcriptase (TERT) through acquired mutations in the TERT promoter. TERT, the catalytically active and rate-limiting subunit of telomerase, functions to maintain telomeres, which cap and protect the ends of chromosomes. Our previous work has demonstrated that the transcription factor GABP - and specifically its tetramer-forming isoform GABPb1L - binds and activates the mutant TERT promoter. The generation of CRISPR-induced indels in GABPb1L results in a gradual loss of cell viability in TERT promoter mutant but not TERT promoter wild type tumor cells in vitro, but the extent to which GABPb1L function is compromised in this setting is unclear. Thus, the potential for use of GABPb1L as an effective therapeutic target for TERT promoter mutant GBM requires further investigation. Here, we use CRISPR-based strategies to demonstrate that full knockout of GABPb1L is rapidly lethal in TERT promoter mutant cells in vitro, in association with a decrease in both TERT mRNA and telomerase activity. Heterozygous deletion of GABPb1L in the context of TERT promoter mutations leads to slowed growth of orthotopic xenograft tumors in mice, and prolonged survival. Additionally, inducible RNAi-mediated inhibition of GABPb1L in growing tumors is also capable of decreasing tumor burden and increasing survival, further strongly suggesting that targeting GABPb1L in patient tumors could be a viable treatment strategy. Finally, reduced GABPb1L synergizes with temozolomide (TMZ) therapy such that TMZ treatment in the context of low GABPb1L and low TERT leads to a complete ablation of orthotopic GBM xenografts. These results highlight the potential to improve disease outcomes by targeting TERT through inhibition of GABPb1L, particularly in conjunction with TMZ treatment.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2019

The Salix psammophila SpRLCK1 involved in drought and salt tolerance

Receptor-like cytoplasmic kinases ( RLCKs ) play critical roles in biotic and abiotic stress responses in plants. However, the functions of RLCKs from the desert shrub willow Salix psammophila have not been characterized. Here, we focused on the biological function of SpRLCK1 , which was previously identified as a potential drought-related gene. Phylogenetic analysis and subcellular localization revealed that SpRLCK1 was a cytoplasmic-localized protein with a protein kinase domain and belonged to the RLCK VIIa subclass. Gene expression profile revealed that SpRLCK1 was predominantly expressed in the root, being consistent with the GUS staining of pSpRLCK1 :GUS transgenic plants. Additionally, the expression of SpRLCK1 was significantly induced by drought and salt stresses. To verify the function of SpRLCK1 , we generated its overexpressing transgenic lines in Arabidopsis thaliana. The SpRLCK1 -overexpressing plants exhibited higher tolerance to drought and salt stresses, as evidenced by the higher survival rate, relative water content and antioxidant enzyme activity than those of wild-type plants. The SpRLCK1 -overexpressing plants enhanced drought and salt tolerance by improving ROS-scavenging activities. A co-expression network for SpRLCK1 was constructed, and the expression analysis indicated that SpRLCK1 regulated the expression of a series of stress-related genes. Ultimately, our results demonstrate that SpRLCK1 confers plant drought and salt tolerance through enhancing the activity of antioxidant enzymes and cooperating with stress-related genes.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2001

Focal adhesion kinase is involved in mechanosensing during fibroblast migration

Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase localized at focal adhesions and is believed to mediate adhesion-stimulated effects. Although ablation of FAK impairs cell movement, it is not clear whether FAK might be involved in the guidance of cell migration, a role consistent with its putative regulatory function. We have transfected FAK-null fibroblasts with FAK gene under the control of the tetracycline repression system. Cells were cultured on flexible polyacrylamide substrates for the detection of traction forces and the application of mechanical stimulation. Compared with control cells expressing wild-type FAK, FAK-null cells showed a decrease in migration speed and directional persistence. In addition, whereas FAK-expressing cells responded to exerted forces by reorienting their movements and forming prominent focal adhesions, FAK-null cells failed to show such responses. Furthermore, FAK-null cells showed impaired responses to decreases in substrate flexibility, which causes control cells to generate weaker traction forces and migrate away from soft substrates. Cells expressing Y397F FAK, which cannot be phosphorylated at a key tyrosine site, showed similar defects in migration pattern and force-induced reorientation as did FAK-null cells. However, other aspects of F397-FAK cells, including the responses to substrate flexibility and the amplification of focal adhesions upon mechanical stimulation, were similar to that of control cells. Our results suggest that FAK plays an important role in the response of migrating cells to mechanical input. In addition, phosphorylation at Tyr-397 is required for some, but not all, of the functions of FAK in cell migration.

NASA Discipline Cell Biology↗

NASA NTRS2002

Parathyroid hormone-dependent signaling pathways regulating genes in bone cells

Parathyroid hormone (PTH) is an 84-amino-acid polypeptide hormone functioning as a major mediator of bone remodeling and as an essential regulator of calcium homeostasis. PTH and PTH-related protein (PTHrP) indirectly activate osteoclasts resulting in increased bone resorption. During this process, PTH changes the phenotype of the osteoblast from a cell involved in bone formation to one directing bone resorption. In addition to these catabolic effects, PTH has been demonstrated to be an anabolic factor in skeletal tissue and in vitro. As a result, PTH has potential medical application to the treatment of osteoporosis, since intermittent administration of PTH stimulates bone formation. Activation of osteoblasts by PTH results in expression of genes important for the degradation of the extracellular matrix, production of growth factors, and stimulation and recruitment of osteoclasts. The ability of PTH to drive changes in gene expression is dependent upon activation of transcription factors such as the activator protein-1 family, RUNX2, and cAMP response element binding protein (CREB). Much of the regulation of these processes by PTH is protein kinase A (PKA)-dependent. However, while PKA is linked to many of the changes in gene expression directed by PTH, PKA activation has been shown to inhibit mitogen-activated protein kinase (MAPK) and proliferation of osteoblasts. It is now known that stimulation of MAPK and proliferation by PTH at low concentrations is protein kinase C (PKC)-dependent in both osteoblastic and kidney cells. Furthermore, PTH has been demonstrated to regulate components of the cell cycle. However, whether this regulation requires PKC and/or extracellular signal-regulated kinases or whether PTH is able to stimulate other components of the cell cycle is unknown. It is possible that stimulation of this signaling pathway by PTH mediates a unique pattern of gene expression resulting in proliferation in osteoblastic and kidney cells; however, specific examples of this are still unknown. This review will focus on what is known about PTH-mediated cell signaling, and discuss the established or putative PTH-regulated pattern of gene expression in osteoblastic cells following treatment with catabolic (high) or anabolic (low) concentrations of the hormone.

NASA Discipline Musculoskeletal↗

DOE OSTI2021

Genome-wide association of the metabolic shifts underpinning dark-induced senescence in Arabidopsis

Dark-induced senescence provokes profound metabolic shifts to recycle nutrients and to guarantee plant survival. To date, research on these processes has largely focused on characterizing mutants deficient in individual pathways. Here, we adopted a time-resolved genome-wide association-based approach to characterize dark-induced senescence by evaluating the photochemical efficiency and content of primary and lipid metabolites at the beginning, or after 3 or 6 days in darkness. We discovered six patterns of metabolic shifts and identified 215 associations with 81 candidate genes being involved in this process. Among these associations, we validated the roles of four genes associated with glycine, galactinol, threonine, and ornithine levels. We also demonstrated the function of threonine and galactinol catabolism during dark-induced senescence. Intriguingly, we determined that the association between tyrosine contents and TYROSINE AMINOTRANSFERASE 1 influences enzyme activity of the encoded protein and transcriptional activity of the gene under normal and dark conditions, respectively. Moreover, the single-nucleotide polymorphisms affecting the expression of THREONINE ALDOLASE 1 and the amino acid transporter gene AVT 1 B, respectively, only underlie the variation in threonine and glycine levels in the dark. Taken together, these results allow us to present a very detailed model of the metabolic aspects of dark-induced senescence, as well as the process itself.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2000

New genes and new biological roles for expansins

Expansins are extracellular proteins that loosen plant cell walls in novel ways. They are thought to function in cell enlargement, pollen tube invasion of the stigma (in grasses), wall disassembly during fruit ripening, abscission and other cell separation events. Expansins are encoded by two multigene families and each gene is often expressed in highly specific locations and cell types. Structural analysis indicates that one expansin region resembles the catalytic domain of family-45 endoglucanases but glucanase activity has not been detected. The genome projects have revealed numerous expansin-related sequences but their putative wall-loosening functions remain to be assessed.

NASA Program Fundamental Space Biology↗

NASA NTRS1996

Towards an understanding of the structural and functional properties of MscL, a mechanosensitive channel in bacteria

Whether it be to sense a touch, arterial pressure, or an osmotic gradient across a cell membrane, essentially all living organisms require the capability of detecting mechanical force. Electrophysiological evidence has suggested that mechanosensitive ion channels play a major role in many systems where mechanical force is detected. But, despite their biological importance, determination of the most basic structural and functional features of mechanosensitive channels has only recently become possible. A gene called mscL, which was isolated from Escherichia coli, was the first gene shown to encode a mechanosensitive channel activity. This channel directly responds to tension in the membrane; no other proteins are required. MscL appears to be a homohexamer of a 136 amino acid polypeptide that is highly alpha helical, contains two transmembrane domains, and has both the amino and carboxyl termini in the cytoplasm. The study of the MscL protein remains, to date, one of the most viable options for understanding the structural and functional characteristics of a mechanosensitive channel.

NASA Discipline Cell Biology↗

NASA NTRS2000

An archaeal genomic signature

Comparisons of complete genome sequences allow the most objective and comprehensive descriptions possible of a lineage's evolution. This communication uses the completed genomes from four major euryarchaeal taxa to define a genomic signature for the Euryarchaeota and, by extension, the Archaea as a whole. The signature is defined in terms of the set of protein-encoding genes found in at least two diverse members of the euryarchaeal taxa that function uniquely within the Archaea; most signature proteins have no recognizable bacterial or eukaryal homologs. By this definition, 351 clusters of signature proteins have been identified. Functions of most proteins in this signature set are currently unknown. At least 70% of the clusters that contain proteins from all the euryarchaeal genomes also have crenarchaeal homologs. This conservative set, which appears refractory to horizontal gene transfer to the Bacteria or the Eukarya, would seem to reflect the significant innovations that were unique and fundamental to the archaeal "design fabric." Genomic protein signature analysis methods may be extended to characterize the evolution of any phylogenetically defined lineage. The complete set of protein clusters for the archaeal genomic signature is presented as supplementary material (see the PNAS web site, www.pnas.org).

Non-NASA Center↗

DOE OSTI2024

Versatile allelic replacement and self-excising integrative vectors for plasmid genome mutation and complementation

ABSTRACT The ability to better understand the function of proteins expressed by bacteria has typically relied upon the development of genetic mutant strains. This approach has been especially challenging for plasmid-encoded genes, as most of the previously described allelic replacement vectors are inefficient for plasmid genome mutation as they either rely on plasmid-derived counterselection toxins or depend on other strategies suchsacB,tetA, andrpsLwhich have been proven to be less efficient for mutant selection. Integrative vectors lack chromophore indicators, thus requiring laborious screening or excision of the vector’s backbone relies on the introduction of a flippase (FLP)-expressing plasmid. The allelic replacement vector, designated here as pDG1, expresses an X-Gal hydrolyzing enzyme (BgaB) that can be used for blue/white screening allowing identification of colonies that integrated and successfully removed the mutagenesis plasmid without a bias for those still carrying it. pDG1 was further improved by including the rhamnose-inducible Tse2 toxin as a potent counterselection system. The efficacy of pDG1 was validated by deleting portions of the plasmid-encoded VirB4/D4 type IV secretion system and aerobactin-synthesizing operons inSalmonella enterica. The integrative vectors, which contain an ΦC31attPsite and genes encoding ΦC31 integrase (int), can seamlessly integrate to target ΦC31attBonSalmonellaplasmids or chromosome. These vectors were improved by insertingbgaBand FLP-encoding genes so that, following integration, most of the vector’s backbone encompassingint,bgaB, and FLP genes can be excised by FLP, without the need for another FLP-expressing vector, creating white colonies carrying a stably integrated target gene. As such, we were able to integrate a 9.3-kb DNA fragment toSalmonellachromosome and flipped out most of the integrated vector in one step, leaving the target fragment in the chromosome. IMPORTANCE In spite of the dissemination of multidrug-resistant plasmids among Gram-negative pathogens, including those carrying virulence genes, vector tools for studying plasmid-born genes are lacking. The allelic replacement vectors can be used to generate plasmid or chromosomal mutations including markless point mutations. This is the first report describing a self-excising integrative vector that can be used as a stable single-copy complementing tool to study medically important pathogens includingin vivostudies without the need for antibiotic selection. Overall, our newly developed vectors can be applied for the assessment of the function of plasmid-encoded genes by specifically creating mutations, moving large operons between plasmids and to/from the chromosome, and complementing phenotypes associated with gene mutation. Furthermore, the vectors express chromophores for the detection of target gene modification or colony isolation, avoiding time-consuming screening procedures.

Microbiology↗

DOE OSTI2021

Mitogen-activated protein kinase 6 negatively regulates secondary wall biosynthesis by modulating MYB46 protein stability in Arabidopsis thaliana

The R2R3-MYB transcription factor MYB46 functions as a master switch for secondary cell wall biosynthesis, ensuring the exquisite expression of the secondary wall biosynthetic genes in the tissues where secondary walls are critical for growth and development. At the same time, suppression of its function is needed when/where formation of secondary walls is not desirable. Little is known about how this opposing control of secondary cell wall formation is achieved. We used both transient and transgenic expression of MYB46 and mitogen-activated protein kinase 6 (MPK6) to investigate the molecular mechanism of the post-translational regulation of MYB46. We show that MYB46 is phosphorylated by MPK6, leading to site specific phosphorylation-dependent degradation of MYB46 by the ubiquitin-mediated proteasome pathway. In addition, the MPK6-mediated MYB46 phosphorylation was found to regulate in planta secondary wall forming function of MYB46. Furthermore, we provide experimental evidences that MYB83, a paralog of MYB46, is not regulated by MPK6. The coupling of MPK signaling to MYB46 function provides insights into the tissue- and/or condition-specific activity of MYB46 for secondary wall biosynthesis.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

The quantitative genetics of gene expression in Mimulus guttatus

Gene expression can be influenced by genetic variants that are closely linked to the expressed gene (cis eQTLs) and variants in other parts of the genome (trans eQTLs). We created a multiparental mapping population by sampling genotypes from a single natural population of Mimulus guttatus and scored gene expression in the leaves of 1,588 plants. We find that nearly every measured gene exhibits cis regulatory variation (91% have FDR < 0.05). cis eQTLs are usually allelic series with three or more functionally distinct alleles. The cis locus explains about two thirds of the standing genetic variance (on average) but varies among genes and tends to be greatest when there is high indel variation in the upstream regulatory region and high nucleotide diversity in the coding sequence. Despite mapping over 10,000 trans eQTL / affected gene pairs, most of the genetic variance generated by trans acting loci remains unexplained. This implies a large reservoir of trans acting genes with subtle or diffuse effects. Mapped trans eQTLs show lower allelic diversity but much higher genetic dominance than cis eQTLs. Several analyses also indicate that trans eQTLs make a substantial contribution to the genetic correlations in expression among different genes. They may thus be essential determinants of “gene expression modules,” which has important implications for the evolution of gene expression and how it is studied by geneticists.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Use of virus‐induced gene silencing to characterize genes involved in modulating hypersensitive cell death in maize

Abstract Plant disease resistance proteins (R‐proteins) detect specific pathogen‐derived molecules, triggering a defence response often including a rapid localized cell death at the point of pathogen penetration called the hypersensitive response (HR). The maize Rp1‐D21 gene encodes a protein that triggers a spontaneous HR causing spots on leaves in the absence of any pathogen. Previously, we used fine mapping and functional analysis in a Nicotiana benthamiana transient expression system to identify and characterize a number of genes associated with variation in Rp1‐D21 ‐induced HR. Here we describe a system for characterizing genes mediating HR, using virus‐induced gene silencing (VIGS) in a maize line carrying Rp1‐D21 . We assess the roles of 12 candidate genes. Three of these genes, SGT1 , RAR1 , and HSP90 , are required for HR induced by a number of R‐proteins across several plant–pathogen systems. We confirmed that maize HSP90 was required for full Rp1‐D21 ‐induced HR. However, suppression of SGT1 expression unexpectedly increased the severity of Rp1‐D21 ‐induced HR while suppression of RAR1 expression had no measurable effect. We confirmed the effects on HR of two genes we had previously validated in the N. benthamiana system, hydroxycinnamoyltransferase and caffeoyl CoA O‐methyltransferase . We further showed the suppression the expression of two previously uncharacterized, candidate genes, IQ calmodulin binding protein ( IQM3 ) and vacuolar protein sorting protein 37 , suppressed Rp1‐D21 ‐induced HR. This approach is an efficient way to characterize the roles of genes modulating the hypersensitive defence response and other dominant lesion phenotypes in maize.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Metals and methylotrophy: Via global gene expression studies

A number of minerals, such as copper, cobalt, and rare earth elements (REE), are essential modulators of microbial one-carbon metabolism. This chapter provides an overview of the gene expression study design and analysis protocols for uncovering REE-induced changes in methylotrophic bacteria. By interrogating relationships and differences in total gene expression induced by mineral micronutrients, a deeper understanding of gene regulation at a systems scale can be gained. With careful design and execution of RNA-sequencing experiments, thorough processing and assessment of read quality can be utilized to assess and adjust for possible biases. Here, by ensuring only quality data are utilized in downstream processes, differential gene expression, overrepresented analyses, and gene-set enrichment analyses provide reliable and reproducible representation of pathways and functions which are being affected by changes in environmental conditions.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Systematic approaches to C-lignin engineering in Medicago truncatula

Abstract Background C-lignin is a homopolymer of caffeyl alcohol present in the seed coats of a variety of plant species including vanilla orchid, various cacti, and the ornamental plant Cleome hassleriana . Because of its unique chemical and physical properties, there is considerable interest in engineering C-lignin into the cell walls of bioenergy crops as a high-value co-product of bioprocessing. We have used information from a transcriptomic analysis of developing C. hassleriana seed coats to suggest strategies for engineering C-lignin in a heterologous system, using hairy roots of the model legume Medicago truncatula . Results We systematically tested strategies for C-lignin engineering using a combination of gene overexpression and RNAi-mediated knockdown in the caffeic acid/5-hydroxy coniferaldehyde 3/5-O-methyltransferase ( comt ) mutant background, monitoring the outcomes by analysis of lignin composition and profiling of monolignol pathway metabolites. In all cases, C-lignin accumulation required strong down-regulation of caffeoyl CoA 3- O -methyltransferase (CCoAOMT) paired with loss of function of COMT. Overexpression of the Selaginella moellendorffii ferulate 5-hydroxylase ( SmF5H ) gene in comt mutant hairy roots resulted in lines that unexpectedly accumulated high levels of S-lignin. Conclusion C-Lignin accumulation of up to 15% of total lignin in lines with the greatest reduction in CCoAOMT expression required the strong down-regulation of both COMT and CCoAOMT, but did not require expression of a heterologous laccase, cinnamyl alcohol dehydrogenase (CAD) or cinnamoyl CoA reductase (CCR) with preference for 3,4-dihydroxy-substituted substrates in M. truncatula hairy roots. Cell wall fractionation studies suggested that the engineered C-units are not present in a heteropolymer with the bulk of the G-lignin.

09 BIOMASS FUELS↗