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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

CsbZIP2-miR9748-CsNPF4.4 Module Mediates High Temperature Tolerance of Cucumber Through Jasmonic Acid Pathway

High temperature stress seriously affects the growth of cucumber seedlings, and even leads to a decline in yield and quality. miRNAs have been shown to be involved in regulating the response to stress in plants, but little is known about its effects on cucumber high temperature stress tolerance. Here, we found that high temperature stress induced the expression of miR9748 in cucumber. Overexpression of cucumber miR9748 in Arabidopsis improved high temperature tolerance. Transcriptome analysis revealed that miR9748 might mediate high temperature tolerance through plant hormone signal pathway. 5′ RNA ligase-mediated rapid amplification of cDNA ends (5′ RLM-RACE) and transient transformation technology demonstrated that CsNPF4.4 was the target gene of miR9748. CsNPF4.4 overexpression plants decreased high temperature tolerance accompanied by reducing the content of jasmonic acid (JA), but alleviated by foliar application of methyl jasmonate, indicating that CsNPF4.4 negatively regulated high temperature stress tolerance through inhibition JA signal pathway. Furthermore, high temperature stress also increased the expression level of CsbZIP2 . Yeast one-hybrid and dual-luciferase assays showed that CsbZIP2 directly bound to the promoter of MIR9748 to induce its expression. Taken together, our results indicated that CsbZIP2 directly regulated miR9748 expression to cleave CsNPF4.4 to mediate high temperature tolerance through JA pathway.

Li, Lan↗
DOE OSTI2023

A transcriptomic atlas of acute stress response to low pH in multiple Issatchenkia orientalis strains

Because of its natural stress tolerance to low pH, Issatchenkia orientalis (a.k.a. Pichia kudriavzevii) is a promising non-model yeast for bio-based production of organic acids. Yet, this organism is relatively unstudied, and specific mechanisms of its tolerance to low pH are poorly understood, limiting commercial use. In this study, we selected 12 I. orientalis strains with varying acid stress tolerance (six tolerant and six susceptible) and profiled their transcriptomes in different pH conditions to study potential mechanisms of pH tolerance in this species. We identified hundreds of genes whose expression response is shared by tolerant strains but not by susceptible strains, or vice versa, as well as genes whose responses are reversed between tolerant and susceptible strains. We mapped regulatory mechanisms of transcriptomic responses via motif analysis as well as differential network reconstruction, identifying several transcription factors, including Stb5, Mac1, and Rtg1/Rtg3, some of which are known for their roles in acid response in Saccharomyces cerevisiae. Functional genomics analysis of short-listed genes and transcription factors suggested significant roles for energy metabolism and translation-related processes, as well as the cell wall integrity pathway and RTG-dependent retrograde signaling pathway. Finally, we conducted additional experiments for two organic acids, 3-hydroxypropionate and citramalate, to eliminate acid-specific effects and found potential roles for glycolysis and trehalose biosynthesis specifically for response to low pH. In summary, our approach of comparative transcriptomics and phenotypic contrasting, along with a multi-pronged bioinformatics analysis, suggests specific mechanisms of tolerance to low pH in I. orientalis that merit further validation through experimental perturbation and engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Data for A Transcriptomic Atlas of Acute Stress Response to Low pH in Multiple Issatchenkia orientalis Strains

Because of its natural stress tolerance to low pH, Issatchenkia orientalis (a.k.a. Pichia kudriavzevii ) is a promising non-model yeast for bio-based production of organic acids. Yet, this organism is relatively unstudied, and specific mechanisms of its tolerance to low pH are poorly understood, limiting commercial use. In this study, we selected 12 I. orientalis strains with varying acid stress tolerance (six tolerant and six susceptible) and profiled their transcriptomes in different pH conditions to study potential mechanisms of pH tolerance in this species. We identified hundreds of genes whose expression response is shared by tolerant strains but not by susceptible strains, or vice versa, as well as genes whose responses are reversed between tolerant and susceptible strains. We mapped regulatory mechanisms of transcriptomic responses via motif analysis as well as differential network reconstruction, identifying several transcription factors, including Stb5, Mac1, and Rtg1/Rtg3, some of which are known for their roles in acid response in Saccharomyces cerevisiae . Functional genomics analysis of short-listed genes and transcription factors suggested significant roles for energy metabolism and translation-related processes, as well as the cell wall integrity pathway and RTG-dependent retrograde signaling pathway. Finally, we conducted additional experiments for two organic acids, 3-hydroxypropionate and citramalate, to eliminate acid-specific effects and found potential roles for glycolysis and trehalose biosynthesis specifically for response to low pH. In summary, our approach of comparative transcriptomics and phenotypic contrasting, along with a multi-pronged bioinformatics analysis, suggests specific mechanisms of tolerance to low pH in I. orientalis that merit further validation through experimental perturbation and engineering.

Conversion↗
NASA NTRS2014

Probiotics in the Space Food System: Delivery, Microgravity Effects, and the Potential Benefit to Crew Health

As mission distance and duration increase, the need grows for non‐invasive disease prevention and immunomodulation, especially given the limited medical response capability expected for these missions and the immune dysregulation documented in crew. Additionally, changes in diet, lifestyle, antibiotic usage, and the environmental stresses during spaceflight may alter crewmembers' intestinal microbiome. The addition of probiotic bacteria to the space food system is expected to confer immunostimulatory benefits on crewmembers, with the potential to counteract the immune dysregulation that has been documented in spaceflight. Based on previous studies that demonstrated unique microbiological responses to the low shear environment of spaceflight, probiotic organisms hold the potential to induce enhanced beneficial responses through mechanisms, such as beneficial interactions with human immune cells and repression of colonization of pathogens on the mucosa. The work presented here will begin to address two research gaps related to providing probiotics in spaceflight: 1) delivery, and 2) the effect of the low shear microgravity environment on probiotic attributes. The probiotic Lactobacillus acidophilus was selected for investigation due to its wide commercial use and documented benefits that include inhibition of virulence related gene expression in pathogens and mucosal stimulation of immune cells. The delivery system for probiotics has not been determined for spaceflight, where the food system is shelf stable and the lack of refrigeration prevents the use of traditional dairy delivery methods. In order to demonstrate the potential of the space food system to deliver viable probiotic bacteria to crewmembers, the probiotic L. acidophilus was packaged in high barrier flight packaging in nonfat dry milk (NFDM) or retained in commercial capsule form. Viable cells were enumerated over 8 months of storage at 22, 4, and ‐80ºC. The survival of L. acidophilus rehydrated in NFDM, in a PBS control, and directly from the capsule was also evaluated following stress challenge with simulated gastric and intestinal juices to determine the method that would deliver the most viable cells to the intestine, where they would be expected to confer beneficial effects. L. acidophilus was found to be stable to gastric and intestinal juice challenge when delivered in rehydrated NFDM, even after two hours of exposure. In comparison, L. acidophilus was reduced by 1‐5 logs when exposed to gastric and intestinal juice directly and when rehydrated in a PBS control. Shelf life data indicated that L. acidophilus would require refrigerated or frozen storage to remain viable at adequate levels over the multi‐year storage periods required for spaceflight. This study suggests that the protective effect provided by the dairy matrix, and not merely rehydration prior to consumption, extends probiotic viability and stress tolerance during storage in spaceflight and in simulated digestion conditions more adequately than a capsule. In addition to effective delivery, it is essential to understand the microgravity effects on the stress tolerances and genetic expression of probiotic bacteria to enable optimization of growth, survival, strain selection, and conferred benefits in spaceflight. In our current study, ground‐based characterization of the growth, stress response, and transcriptomic response of L. acidophilus will be accomplished using the low shear modeled microgravity (LSMMG) culture environment in the rotating wall vessel (RWV). We hypothesize that L. acidophilus will maintain or improve its growth and stress response following culture in the LSMMG environment, and that transcriptomic analysis will define the associated molecular mechanism(s), resulting in the ability to optimize strain selection.

Castro, S. L.↗
DOE OSTI2025

Breeding of microbiomes conferring salt tolerance to plants

Microbiome breeding through host-mediated selection is a technique to artificially select for microbiomes conferring beneficial properties to plants. Using a systematic selection protocol that maximises the heritability of microbiome effects, transmission fidelity, and microbiome stability through multiple selection cycles, we previously developed root-associated microbial communities conferring sodium and aluminium tolerance to Brachypodium distachyon, a model for cereal crops. Here, we explore the physiological mechanisms underlying our selected microbiomes’ effect on plant fitness and analyse how our selection protocol shaped the composition and structure of these microbiomes. We analysed the effects of our selected microbiomes on plant fitness and tissue-nutrient concentration, then used 16S rRNA amplicon sequencing to examine microbial community composition and co-occurrence network patterns. Our sodium-selected microbiomes reduced leaf sodium concentration by ~ 50%, whereas the aluminium-selected microbiomes had no effect on leaf-tissue nutrient concentration, suggesting different mechanisms underlying the microbiome-mediated stress tolerance. By testing the selected microbiomes in a cross-fostering experiment, we show that our artificially selected microbiomes attained (a) ecological robustness contributing to transplantability (i.e. inheritance) of microbiome-encoded effects between plants; and (b) network features identifying key bacteria promoting salt-stress tolerance. Combined, these findings elucidate critical mechanisms underlying host-mediated artificial selection as a framework to breed microbiomes with targeted benefits for plants under salt stresses, with significant implications for sustainable agriculture.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Dissecting the metabolic reprogramming of maize root under nitrogen-deficient stress conditions

The growth and development of maize (Zea mays L.) largely depends on its nutrient uptake through the root. Hence, studying its growth, response, and associated metabolic reprogramming to stress conditions is becoming an important research direction. A genome-scale metabolic model (GSM) for the maize root was developed to study its metabolic reprogramming under nitrogen stress conditions. The model was reconstructed based on the available information from KEGG, UniProt, and MaizeCyc. Transcriptomics data derived from the roots of hydroponically grown maize plants were used to incorporate regulatory constraints in the model and simulate nitrogen-non-limiting (N+) and nitrogen-deficient (N-) condition. Model-predicted flux-sum variability analysis achieved 70% accuracy compared with the experimental change of metabolite levels. In addition to predicting important metabolic reprogramming in central carbon, fatty acid, amino acid, and other secondary metabolism, maize root GSM predicted several metabolites (l-methionine, l-asparagine, l-lysine, cholesterol, and l-pipecolate) playing a regulatory role in the root biomass growth. Furthermore, this study revealed eight phosphatidylcholine and phosphatidylglycerol metabolites which, even though not coupled with biomass production, played a key role in the increased biomass production under N-deficient conditions. Overall, the omics-integrated GSM provides a promising tool to facilitate stress condition analysis for maize root and engineer better stress-tolerant maize genotypes.

Plant Sciences↗
DOE OSTI2022

Novel Mutant Alleles Reveal a Role of the Extra-Large G Protein in Rice Grain Filling, Panicle Architecture, Plant Growth, and Disease Resistance

Plant growth and grain filling are the key agronomical traits for grain weight and yield of rice. The continuous improvement in rice yield is required for a future sustainable global economy and food security. The heterotrimeric G protein complex containing a canonical α subunit (RGA1) couples extracellular signals perceived by receptors to modulate cell function including plant development and grain weight. We hypothesized that, besides RGA1, three atypical, extra-large GTP-binding protein (XLG) subunits also regulate panicle architecture, plant growth, development, grain weight, and disease resistance. Here, we identified a role of XLGs in agronomic traits and stress tolerance by genetically ablating all three rice XLGs individually and in combination using the CRISPR/Cas9 genome editing in rice. For this study, eight (three single, two double, and three triple) null mutants were selected. Three XLG proteins combinatorically regulate seed filling, because loss confers a decrease in grain weight from 14% with loss of one XLG and loss of three to 32% decrease in grain weight. Null mutations in XLG2 and XLG4 increase grain size. The mutants showed significantly reduced panicle length and number per plant including lesser number of grains per panicle compared to the controls. Loss-of-function of all individual XLGs contributed to 9% more aerial biomass compared to wild type (WT). The double mutant showed improved salinity tolerance. Moreover, loss of the XLG gene family confers hypersensitivity to pathogens. Our findings suggest that the non-canonical XLGs play important roles in regulating rice plant growth, grain filling, panicle phenotype, stress tolerance, and disease resistance. Genetic manipulation of XLG s has the potential to improve agronomic properties in rice.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Improved Biofuel Production through Discovery and Engineering of Terpene Metabolism in Switchgrass

Project Objectives - Of the myriad specialized metabolites that plants deploy to adapt to environmental challenges, terpenes form the largest group. In many major crops, unique terpene blends serve as key stress defenses that directly impact plant fitness and yield. In addition, terpenes, such as bisabolene and pinene, are used for producing renewable biofuels. Essential to advancing a broader use of terpenes for biofuel feedstock engineering is a system-wide knowledge of the diverse biosynthetic machinery and defensive potential of often species-specific terpene blends. The proposed project would merge genome-wide enzyme discovery with comparative –omics, protein structural and plant microbiome studies to define the biosynthesis and stress-defensive functions of the switchgrass (Panicum virgatum) terpene network. These insights would be combined with developing and applying non-transgenic genome editing tools to design plants with desirable terpene blends for higher productivity and biofuel production on marginal lands. As a dedicated lignocellulosic feedstock for U.S. biofuel production with high net energy yield, stress tolerance, and available genome resources, switchgrass is well-suited for devising new avenues for biofuel production. Project Description – The diversity of plant terpene defenses is governed by species-specific families of terpene synthase (TPS) and cytochrome P450 monooxygenase (P450) enzymes. Mining of the switchgrass genome (genotype Alamo) identified ~100 TPS and P450 candidate genes, and combinatorial biochemical analysis of synthesized TPSs and P450s revealed more than a dozen enzymes with common and novel activities. In addition, several identified terpene metabolites and the corresponding transcripts were up-regulated in response to abiotic stressors. These findings demonstrate a unique switchgrass terpene network with probable importance to abiotic stress tolerance, thus providing a large chemical portfolio for optimizing crop resistance, yield, and biofuel composition. Leveraging these preliminary data, we propose to generate a genome-wide map of the switchgrass terpene metabolic network through multi-gene co-expression analyses that allow the efficient cross-validation of TPS and P450 functions. Key enzymes would further be applied to structure-function studies via X-ray protein crystallography, homology modeling and site-directed mutagenesis to gain mechanistic insight into the catalytic specificity of switchgrass terpene metabolism and provide gene and amino acid targets for genome editing. In tandem with terpene pathway discovery, system-wide metabolomics, transcriptomics and proteomics studies in switchgrass accessions of contrasting drought tolerance would define the role of switchgrass terpene metabolism in conferring abiotic stress resilience. Metabolic changes would be assessed in a combined approach of targeted (terpenes) and untargeted metabolite profiling using a high-resolution LC-MS/MS approach, differential gene expression analyses through multiplexed Illumina RNA sequencing, and quantitative analysis of high-priority pathway enzymes using multiple reaction monitoring (MRM). Drawing on these insights, knock-down/out mutants of stress-associated pathway nodes would be generated by optimizing transient virus-induced gene silencing (VIGS) and CRISPR/Cas9 systems under control of the Tobacco Rattle Virus (TRV). The resulting mutant lines would then be analyzed for stress susceptibility and the impact on the root microbiome to define gene functions in planta. Knowledge of terpene pathways, enzyme mechanisms and bioactivities would be applied to enhance switchgrass stress resilience and to tailor-make terpene blends for biofuel production. Here, TRV-enabled CRISPR/Cas9 genome editing, including allele-specific knock-out of redundant genes, engineering of enzyme specificity via structure-guided point mutations, and overexpression of terpene genes relevant to stress-protection or biofuel production, would be used to increase metabolic flux toward desired pathways. Broader Impacts - Integrating the system-wide discovery, mechanistic analysis and non-transgenic genome engineering of the switchgrass terpene network aligns the required steps to unlock the chemical potential of this important metabolite class to generate crops that are more resistant to stress and provide advanced biofuel production in light of rising climate pressures as foreseeable challenges for bioenergy crop cultivation. The proposed project would further offer interdisciplinary student training through active involvement in the project and integration of research concepts and outcomes into newly-developed graduate and undergraduate courses on Plant Biotechnology.

09 BIOMASS FUELS↗
DOE OSTI2023

Identifying biomass yield potential of tetra, hexa, and octoploid prairie cordgrass populations grown on marginal lands

As the demand for renewable energy sources with reduced carbon emissions to the atmosphere increases, bioenergy crops remain an attractive alternative to fossil fuels. Another appeal of bioenergy crops is the potential utilization of marginal lands not suitable for food crops. Prairie cordgrass is a perennial warm season polyploid grass species being considered as a bioenergy crop that also exhibits a high tolerance for salinity and flooding. The ploidy level of prairie cordgrass has previously been suggested to correlate with agronomic traits. However, little is known about the agricultural performance of prairie cordgrass on marginal lands and if a correlation of ploidy level to stress tolerance exists. In this experiment, the productivity of tetraploids, hexaploids, and octoploids was investigated when grown on three different marginal sites including waterlogged, saline, and low-nutrient gravelly soils. Here we found that prairie cordgrass populations annually produced dry biomass on a wet marginal site comparable to normal cropland. However, the productivity was remarkedly decreased at both a salt-affected site and a limestone mine reclamation site. We were unable to conclude that different ploidy levels affected the biomass yield of prairie cordgrass grown under abiotic stress. However, there was a differential response of populations to various types of marginal sites. Finally, these results indicate potential genetic resources for developing breeding targets and identifying mechanisms of abiotic stress tolerance. Moreover, we highlight the need to generate diverse ploidy levels of prairie cordgrass in the same genetic backgrounds for future testing.

09 BIOMASS FUELS↗
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↗
DOE OSTI2026

Nutrient limitation shapes functional traits of mycorrhizal fungi and phosphorus-cycling bacteria across an elevation gradient

In nutrient-limited high-elevation ecosystems, plants rely on arbuscular mycorrhizal (AM) fungi to provide mineral phosphorus (P) in the form of phosphate (PO43-). AM fungi gather these nutrients from phosphorus-cycling bacteria (PCBs) that can mineralize PO43- from organic matter and solubilize mineral-bound P. How climate, soil factors, and nutrient limitation influence AM fungi and PCB assembly remains unclear. We collected soil from montane meadows across a 1,000-m elevation gradient on three replicate mountainsides and analyzed AM fungal marker genes, P-cycling genes from shotgun metagenomes, and edaphic measurements. High-elevation soils had nearly 50-fold less soil PO₄³⁻ and 60% more AM fungal hyphae than low-elevation soils. AM fungal turnover was linked to changes in pH, organic carbon, and PO₄³-. The composition of 198 P-cycling genes was influenced by the AM fungal community structure. Drivers of individual PCB functional genes, including pH and organic carbon, varied with gene phylogeny. We found a trade-off in P-cycling strategies across elevation: P-rich, low-elevation soils supported root-colonizing AM fungi and organic P-mineralizing bacteria. P-poor, high-elevation soils were dominated by stress-tolerant AM fungi and mineral P-solubilizing bacteria. Our results suggest that AM fungi and PCB community turnover across elevation are both shaped by pH, organic carbon, and P availability. With continued climate warming, the structure and function of mountaintop ecosystems might shift to resemble lower elevations, disrupting long-established and specialized microbial assemblages, with consequences for P-cycling dynamics and the total P available to plant communities.IMPORTANCEPhosphorus (P) limits plant productivity in high-elevation ecosystems, yet the microbial networks that mobilize P, including arbuscular mycorrhizal (AM) fungi and phosphorus-cycling bacteria (PCBs), remain under-characterized in these nutrient-poor soils. We show that across a 10,00-m elevation gradient, AM fungi and P-cycling gene assemblages shift predictably with pH, organic carbon, and phosphate availability. Higher elevations, with less available P, select for stress-tolerant AM fungal taxa and PCB strategies geared toward mineral solubilization, while low-elevation sites favor root colonization by AM fungi and organic P mineralization. These results suggest that nutrient limitation can constrain microbial community assembly in consistent ways across landscapes. High mountain soils are low in P and rely on a network of underground AM fungi and PCB to deliver nutrients to plants. This study shows how those underground relationships reorganize with elevation and how climate change could collapse long-standing microbial strategies by pushing high-elevation ecosystems toward lowland conditions. As soils warm and dry, the microbial scaffolding that supports alpine plant life may become increasingly unstable.

arbuscular mycorrhizal fungi↗
DOE OSTI2024

Potential for functional divergence in ectomycorrhizal fungal communities across a precipitation gradient

Abstract Functional traits influence the assembly of microbial communities, but identifying these traits in the environment has remained challenging. We studied ectomycorrhizal fungal (EMF) communities inhabiting Populus trichocarpa roots distributed across a precipitation gradient in the Pacific Northwest, USA. We profiled these communities using taxonomic (meta-barcoding) and functional (metagenomic) approaches. We hypothesized that genes involved in fungal drought-stress tolerance and fungal mediated plant water uptake would be most abundant in drier soils. We were unable to detect support for this hypothesis; instead, the abundance of genes involved in melanin synthesis, hydrophobins, aquaporins, trehalose-synthases, and other gene families exhibited no significant shifts across the gradient. Finally, we studied variation in sequence homology for certain genes, finding that fungal communities in dry soils are composed of distinct aquaporin and hydrophobin gene sequences. Altogether, our results suggest that while EMF communities exhibit significant compositional shifts across this gradient, coupled functional turnover, at least as inferred using community metagenomics is limited. Accordingly, the consequences of these distinct EMF communities on plant water uptake remain critically unknown, and future studies targeting the expression of genes involved in drought stress tolerance are required.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Drought and plant litter chemistry alter microbial gene expression and metabolite production

Drought represents a significant stress to microorganisms and is known to reduce microbial activity and organic matter decomposition in Mediterranean ecosystems. However, we lack a detailed understanding of the drought stress response of microbial decomposers. Here we present metatranscriptomic and metabolomic data on the physiological response of in situ microbial communities on plant litter to long-term drought in Californian grass and shrub ecosystems. We hypothesised that drought causes greater microbial allocation to stress tolerance relative to growth pathways. In grass litter, communities from the decade-long ambient and reduced precipitation treatments had distinct taxonomic and functional profiles. The most discernable physiological signatures of drought were production or uptake of compatible solutes to maintain cellular osmotic balance, and synthesis of capsular and extracellular polymeric substances as a mechanism to retain water. The results show a clear functional response to drought in grass litter communities with greater allocation to survival relative to growth that could affect decomposition under drought. In contrast, communities on chemically more diverse and complex shrub litter had smaller physiological differences in response to long-term drought but higher investment in resource acquisition traits across precipitation treatments, suggesting that the functional response to drought is constrained by substrate quality. Our findings suggest, for the first time in a field setting, a trade off between microbial drought stress tolerance, resource acquisition and growth traits in plant litter microbial communities.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

CRISPR-mediated multigene integration enables Shikimate pathway refactoring for enhanced 2-phenylethanol biosynthesis in Kluyveromyces marxianus

Background: 2-phenylethanol (2-PE) is a rose-scented flavor and fragrance compound that is used in food, beverages, and personal care products. Compatibility with gasoline also makes it a potential biofuel or fuel additive. A biochemical process converting glucose or other fermentable sugars to 2-PE can potentially provide a more sustainable and economical production route than current methods that use chemical synthesis and/or isolation from plant material. Results: We work toward this goal by engineering the Shikimate and Ehrlich pathways in the stress-tolerant yeast Kluyveromyces marxianus. First, we develop a multigene integration tool that uses CRISPR-Cas9 induced breaks on the genome as a selection for the one-step integration of an insert that encodes one, two, or three gene expression cassettes. Integration of a 5-kbp insert containing three overexpression cassettes successfully occurs with an efficiency of 51 ± 9% at the ABZ1 locus and was used to create a library of K. marxianus CBS 6556 strains with refactored Shikimate pathway genes. The 3 3 -factorial library includes all combinations of KmARO4, KmARO7, and KmPHA2, each driven by three different promoters that span a wide expression range. Analysis of the refactored pathway library reveals that high expression of the tyrosine-deregulated KmARO4 K221L and native KmPHA2, with the medium expression of feedback insensitive KmARO7 G141S , results in the highest increase in 2-PE biosynthesis, producing 684 ± 73 mg/L. Ehrlich pathway engineering by overexpression of KmARO10 and disruption of KmEAT1 further increases 2-PE production to 766 ± 6 mg/L. The best strain achieves 1943 ± 63 mg/L 2-PE after 120 h fed-batch operation in shake flask cultures. Conclusions: The CRISPR-mediated multigene integration system expands the genome-editing toolset for K. marxianus, a promising multi-stress tolerant host for the biosynthesis of 2-PE and other aromatic compounds derived from the Shikimate pathway.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Transcriptional Regulatory Networks Involved in C3–C4 Alcohol Stress Response and Tolerance in Yeast

Alcohol toxicity significantly impacts the titer and productivity of industrially produced biofuels. To overcome this limitation, we must find and use strategies to improve stress tolerance in production strains. Previously, we developed a multiplex navigation of a global regulatory network (MINR) library that targeted 25 regulatory genes that are predicted to modify global regulation in yeast under different stress conditions. Herein, we expanded this concept to target the active sites of 47 transcriptional regulators using a saturation mutagenesis library. The 47 targeted regulators interact with more than half of all yeast genes. We then screened and selected for C3–C4 alcohol tolerance. We identified specific mutants that have resistance to isopropanol and isobutanol. Notably, the WAR1_K110N variant improved tolerance to both isopropanol and isobutanol. In addition, we investigated the mechanisms for improvement of isopropanol and isobutanol stress tolerance and found that genes related to glycolysis play a role in tolerance to isobutanol, while changes in ATP synthesis and mitochondrial respiration play a role in tolerance to both isobutanol and isopropanol. Overall, this work sheds light on basic mechanisms for isopropanol and isobutanol toxicity and demonstrates a promising strategy to improve tolerance to C3–C4 alcohols by perturbing the transcriptional regulatory network.

09 BIOMASS FUELS↗
DOE OSTI2023

RB-TnSeq identifies genetic targets for improved tolerance of Pseudomonas putida towards compounds relevant to lignin conversion

We report lignin-derived mixtures intended for bioconversion commonly contain high concentrations of aromatic acids, aliphatic acids, and salts. The inherent toxicity of these chemicals places a significant bottleneck upon the effective use of microbial systems for the valorization of these mixtures. Pseudomonas putida KT2440 can tolerate stressful quantities of several lignin-related compounds, making this bacterium a promising host for converting these chemicals to valuable bioproducts. Nonetheless, further increasing P. putida tolerance to chemicals in lignin-rich substrates has the potential to improve bioprocess performance. Accordingly, we employed random barcoded transposon insertion sequencing (RB-TnSeq) to reveal genetic determinants in P. putida KT2440 that influence stress outcomes during exposure to representative constituents found in lignin-rich process streams. The fitness information obtained from the RB-TnSeq experiments informed engineering of strains via deletion or constitutive expression of several genes. Namely, ΔgacAS, ΔfleQ, ΔlapAB, ΔttgR::P tac :ttgABC, Ptac:PP_1150:PP_1152, ..delta..relA, and ΔPP_1430 mutants showed growth improvement in the presence of single compounds, and some also exhibited greater tolerance when grown using a complex chemical mixture representative of a lignin-rich chemical stream. Overall, this work demonstrates the successful implementation of a genome-scale screening tool for the identification of genes influencing stress tolerance against notable compounds within lignin-enriched chemical streams, and the genetic targets identified herein offer promising engineering targets for improving feedstock tolerance in lignin valorization strains of P. putida KT2440.

09 BIOMASS FUELS↗
DOE OSTI2020

COST1 regulates autophagy to control plant drought tolerance

Plants balance their competing requirements for growth and stress tolerance via a sophisticated regulatory circuitry that controls responses to the external environments. We have identified a plant-specific gene, COST1 (constitutively stressed 1), that is required for normal plant growth but negatively regulates drought resistance by influencing the autophagy pathway. An Arabidopsis thaliana cost1 mutant has decreased growth and increased drought tolerance, together with constitutive autophagy and increased expression of drought-response genes, while overexpression of COST1 confers drought hypersensitivity and reduced autophagy. The COST1 protein is degraded upon plant dehydration, and this degradation is reduced upon treatment with inhibitors of the 26S proteasome or autophagy pathways. The drought resistance of a cost1 mutant is dependent on an active autophagy pathway, but independent of other known drought signaling pathways, indicating that COST1 acts through regulation of autophagy. In addition, COST1 colocalizes to autophagosomes with the autophagosome marker ATG8e and the autophagy adaptor NBR1, and affects the level of ATG8e protein through physical interaction with ATG8e, indicating a pivotal role in direct regulation of autophagy. We propose a model in which COST1 represses autophagy under optimal conditions, thus allowing plant growth. Under drought, COST1 is degraded, enabling activation of autophagy and suppression of growth to enhance drought tolerance. Our research places COST1 as an important regulator controlling the balance between growth and stress responses via the direct regulation of autophagy.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Enhanced Salt Tolerance of Torreya grandis Genders Is Related to Nitric Oxide Level and Antioxidant Capacity

Nitric oxide (NO), a bioactive molecule, is often involved in the regulation of physiological and biochemical processes in stressed plants. However, the effects of NO donors on dioecious plants remain unclear. Using a pot experiment, female and male Torreya grandis were used to study the role of sex and NO in salt stress tolerance. In the present study, female and male T. grandis seedlings pretreated with an NO donor (sodium nitroprusside, SNP) were exposed to salt stress, and then leaf relative water content (RWC), photosynthetic pigments, chlorophyll fluorescence parameters, NO and glutathione levels, oxidative damage, and antioxidant enzyme activities were investigated. Female T. grandis plants had better tolerance to salinity, as they were characterized by significantly higher RWC, pigment content, and photochemical activities of photosystem II (PSII) and fewer negative effects associated with higher nitrate reductase (NR) activity and NO content. Pretreatment with an NO donor further increased the endogenous NO content and NR activity of both female and male T. grandis plants compared with salt treatment. Moreover, pretreatment with an NO donor alleviated salt-induced oxidative damage of T. grandis , especially in male plants, as indicated by reduced lipid peroxidation, through an enhanced antioxidant system, including proline and glutathione accumulation, and increased antioxidant enzyme activities. However, the ameliorating effect of the NO donor was not effective in the presence of the NO scavenger (Nω-nitro-L-arginine methyl ester, L-name). In conclusion, enhanced salt tolerance in T. grandis plants is related to nitric oxide levels and the supply of NO donors is an interesting strategy for alleviating the negative effect of salt on T. grandis . Our data provide new evidence to contribute to the current understanding of NO-induced salt stress tolerance.

Liu, Yang↗