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DOE OSTI2018

Functional FRIGIDA allele enhances drought tolerance by regulating the P5CS1 pathway in Arabidopsis thaliana

Highlights: • ColFRI plants increase drought resistance. • FRI positively upregulates P5CS1 expression during drought tolerance. • The effect of FRI on drought resistance is dependent on FLC gene. Flowering at the right time is important for the reproductive success of plants and their response to environmental stress. In Arabidopsis, a major determinant of natural variation in flowering time is FRIGIDA (FRI). In the present study, we show that overexpression of the functional FRIGIDA gene in wild-type Col background (ColFRI) positively enhances the drought tolerance by activating P5CS1 expression and promoting proline accumulation during water stress. Furthermore, no significant changes in FRI gene and protein expression levels were observed with drought treatment, whereas P5CS1 protein expression significantly increased. In contrast, vernalization treatment efficiently reduced P5CS1 expression levels and resulted in a decrease in drought tolerance in the ColFRI plants. The flc mutants with a functional FRI background also relieved FRI-mediated activation of P5CS1 during drought tolerance. Taken together, our findings reveal the novel function of FRI in enhancing drought resistance through its downstream P5CS1 pathway during water-deficit stress, which is dependent on its target, the FLC gene.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Overexpression of a novel transcriptional repressor GmMYB3a negatively regulates salt–alkali tolerance and stress-related genes in soybean

Highlights: • We identified and characterized GmMYB3a of R2R3 myeloblastosis repressor family. • GmMYB3a is induced by various abiotic stresses. • For this study, we used a transgenic soybean over-expressing GmMYB3a. • We evaluated the photosynthesis index under salt-alkali treatments. • GmMYB3a was found to negatively affect the response of plants to salt stress. Myeloblastosis (MYB) transcription factor (TF) plays a positive role in the growth and stress response of plants; however, information on the functions of MYB repressors in soybean is limited. In the present study, the gene GmMYB3a was identified and characterized as a member of the R2R3 MYB repressor family, which is induced by various abiotic stresses. To understand the functions of GmMYB3a, a transgenic soybean over-expressing GmMYB3a was obtained and the photosynthetic index under salt–alkali treatments was evaluated. The transgenic line exhibited a series of negative regulation relative to the wild-type control. Quantitative real time polymerase chain reaction revealed that the physiological parameters, including soluble sugar, free proline, and chlorophyll contents; and photosynthetic rate decreased in the transgenic plants. Furthermore, GmMYB3a overexpression down-regulated a set of key genes associated with plant defense signal pathways. These finding suggest that GmMYB3a negatively affects the response of plants to salt stress.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Biophysical characterization of actin bundles generated by the Chlamydia trachomatis Tarp effector

Highlights: • The F-actin binding domain of Tarp is sufficient to bundle filamentous actin. • Actin bundles formed by Tarp are more flexible than that of α-actinin and fascin. • Tarp mediated actin bundling is independent of actin nucleation. Chlamydia trachomatis entry into host cells is mediated by pathogen-directed remodeling of the actin cytoskeleton. The chlamydial type III secreted effector, translocated actin recruiting phosphoprotein (Tarp), has been implicated in the recruitment of actin to the site of internalization. Tarp harbors G-actin binding and proline rich domains required for Tarp-mediated actin nucleation as well as unique F-actin binding domains implicated in the formation of actin bundles. Little is known about the mechanical properties of actin bundles generated by Tarp or the mechanism by which Tarp mediates actin bundle formation. In order to characterize the actin bundles and elucidate the role of different Tarp domains in the bundling process, purified Tarp effectors and Tarp truncation mutants were analyzed using Total Internal Reflection Fluorescence (TIRF) microscopy. Our data indicate that Tarp mediated actin bundling is independent of actin nucleation and the F-actin binding domains are sufficient to bundle actin filaments. Additionally, Tarp-mediated actin bundles demonstrate distinct bending stiffness compared to those crosslinked by the well characterized actin bundling proteins fascin and alpha-actinin, suggesting Tarp may employ a novel actin bundling strategy. The capacity of the Tarp effector to generate novel actin bundles likely contributes to chlamydia's efficient mechanism of entry into human cells.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Deletion of the GCW13 gene derepresses Gap1-dependent uptake of amino acids in Pichia pastoris grown on methanol as the sole carbon source

Highlights: • Deletion of GCW13 results in an increased growth rate in P. pastoris GS115 on methanol as the sole carbon source. • The intracellular concentrations of various amino acids are increased in the GCW13-deletion P. pastoris strain. • Gcw13 facilitates the endocytosis of Gap1 in P. pastoris GS115 on methanol as the sole carbon source. In Pichia pastoris, most of the Glycosylphosphatidylinositol (GPI)-anchored proteins are of unknown function. Gcw13, one of these GPI-anchored proteins, was found to exert an inhibitory effect on the growth of the histidine auxotrophic P. pastoris strain GS115 on methanol as the sole carbon source. To investigate the biological function of Gcw13, RNA sequencing (RNA-Seq) was performed to compare the difference of gene expression between GS115 and GCW13-deletion strain D13. RNA-Seq analysis showed that, in strain D13, the expression of genes involved in the methanol utilization pathway or peroxisome biogenesis was not changed, and a high proportion of genes involved in the biosynthesis of amino acids were down-regulated, whereas GAP1, which encodes a general amino acid permease, was significantly up-regulated. Besides, the intracellular concentrations of various amino acids were significantly higher in D13 than that in GS115. We also observed that deletion of GCW13 resulted in more Gap1 presented on the cell surface and more active uptake of the toxic proline analogue l-azetidine-2-carboxylate acid (AzC). These results suggest that Gcw13 suppresses the expression of GAP1 and facilitates the endocytosis of Gap1 on methanol, resulting in decreasing Gap1-dependent uptake of amino acids in P. pastoris, which might contribute to the poor growth of GS115 on methanol.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2017

Structure of thrombospondin type 3 repeats in bacterial outer membrane protein A reveals its intra-repeat disulfide bond-dependent calcium-binding capability

Eukaryotic thrombospondin type 3 repeat (TT3R) is an efficient calcium ion (Ca2+) binding motif only found in mammalian thrombospondin family. TT3R has also been found in prokaryotic cellulase Cel5G, which was thought to forfeit the Ca2+-binding capability due to the formation of intra-repeat disulfide bonds, instead of the inter-repeat ones possessed by eukaryotic TT3Rs. In this study, we have identified an enormous number of prokaryotic TT3R-containing proteins belonging to several different protein families, including outer membrane protein A (OmpA), an important structural protein connecting the outer membrane and the periplasmic peptidoglycan layer in gram-negative bacteria. Here, we report the crystal structure of the periplasmic region of OmpA from Capnocytophaga gingivalis, which contains a linker region comprising five consecutive TT3Rs. The structure of OmpA-TT3R exhibits a well-ordered architecture organized around two tightly-coordinated Ca2+ and confirms the presence of abnormal intra-repeat disulfide bonds. Further mutagenesis studies showed that the Ca2+-binding capability of OmpA-TT3R is indeed dependent on the proper formation of intra-repeat disulfide bonds, which help to fix a conserved glycine residue at its proper position for Ca2+ coordination. Additionally, despite lacking inter repeat disulfide bonds, the interfaces between adjacent OmpA-TT3Rs are enhanced by both hydrophobic and conserved aromatic-proline interactions.

Aromatic-proline interaction↗

DOE OSTI2021

CO 2 -selective membranes containing amino acid salts for CO 2 /N 2 separation

In this paper, facilitated transport membranes comprising polyvinylamine (PVAm) as fixed carrier and different amino acid salts (AAS) as mobile carriers were synthesized for post-combustion capture. The AAS carriers were prepared by deprotonating alanine (Ala), lysine (Lys), and proline (Pro) with 2-(1-piperazinyl)ethylamine (PZEA). CO 2 separation performances of the membranes with these AAS carriers were compared, and the AAS effectiveness to facilitate transport of CO 2 was in the order of PZEA-Pro > PZEA-Lys > PZEA-Ala. Here, the membrane comprising 35 wt.% PVAm and 65 wt.% PZEA-Pro at 57 °C rendered a promising CO 2 permeance of 936 GPU and a CO 2 /N 2 mixed gas selectivity of 193. When the PVAm was reduced to 15 wt.%, i.e., its 20 wt.% was replaced by the mobile carrier of PZEA-Sar (sarcosinate), the permeance further improved to 947 GPU with a remarkable CO 2 /N 2 selectivity of 210. Moreover, thermal gravimetric analysis showed a good thermal stability of the membrane, and membrane stability testing also gave stable transport performance. Furthermore, spectroscopic ellipsometry analysis exhibited a uniform membrane selective layer and an excellent agreement on the membrane thickness of ca. 170 nm measured independently by SEM. In addition, the membranes presented in this paper surpassed both the Robeson upper bound and the modified upper bound, indicating a great potential for CO 2 capture.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2018

Metabolic profiling associated with autophagy of human placenta-derived mesenchymal stem cells by chemical isotope labeling LC−MS

Autophagy has been reported to have a pivotal role in maintaining stemness, regulating immunomodulation and enhancing the survival of mesenchymal stem cells (MSCs). However, the effect of autophagy on MSC metabolism is largely unknown. Here, we report a workflow for examining the impact of autophagy on human placenta-derived MSC (hPMSC) metabolome profiling with chemical isotope labeling (CIL) LC−MS. Rapamycin or 3-methyladenine was successfully used to induce or inhibit autophagy, respectively. Then, {sup 12}C- and {sup 13}C-dansylation labeling LC−MS were used to profile the amine/phenol submetabolome. A total of 935 peak pairs were detected and 50 metabolites were positively identified using the dansylation metabolite standards library, and 669 metabolites were putatively identified based on an accurate mass match in metabolome databases. {sup 12}C/{sup 13}C-p-dimethylaminophenacyl bromide labeling LC−MS was used to analyze the carboxylic acid submetabolome; 4736 peak pairs were detected, among which 33 metabolites were positively identified in the dimethylaminophenacyl metabolite standards library, and 3007 metabolites were putatively identified. PCA/OPLS-DA analysis combined with volcano plots and Venn diagrams was used to determine the significant metabolites. Metabolites pathway analysis demonstrated that hPMSCs appeared to generate more ornithine with the arginine and proline metabolism pathway and utilized more pantothenic acid to synthesize acetyl-CoA in the beta-alanine metabolism pathway when autophagy was activated. Meanwhile, acetyl-CoA conversion to fatty acids led to accumulation in the fatty acid biosynthesis pathway. In contrast, when autophagy was suppressed, a reduction in metabolites demonstrated weakened metabolic activity in these metabolic pathways. Our research provides a more comprehensive understanding of hPMSC metabolism associated with autophagy.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2019

Structural determinants for accurate dephosphorylation of RNA polymerase II by its cognate C-terminal domain (CTD) phosphatase during eukaryotic transcription

The C-terminal domain (CTD) of RNA polymerase II contains a repetitive heptad sequence (YSPTSPS) whose phosphorylation states coordinate eukaryotic transcription by recruiting protein regulators. The precise placement and removal of phosphate groups on specific residues of the CTD are critical for the fidelity and effectiveness of RNA polymerase II–mediated transcription. During transcriptional elongation, phosphoryl-Ser 5 (pSer 5 ) is gradually dephosphorylated by CTD phosphatases, whereas Ser 2 phosphorylation accumulates. Using MS, X-ray crystallography, protein engineering, and immunoblotting analyses, in this work we investigated the structure and function of SSU72 homolog, RNA polymerase II CTD phosphatase (Ssu72, from Drosophila melanogaster ), an essential CTD phosphatase that dephosphorylates pSer 5 at the transition from elongation to termination, to determine the mechanism by which Ssu72 distinguishes the highly similar pSer 2 and pSer 5 CTDs. We found that Ssu72 dephosphorylates pSer 5 effectively but only has low activities toward pSer 7 and pSer 2 . The structural analysis revealed that Ssu72 requires that the proline residue in the substrate's SP motif is in the cis configuration, forming a tight β-turn for recognition by Ssu72. We also noted that residues flanking the SP motif, such as the bulky Tyr 1 next to Ser 2 , prevent the formation of such configuration and enable Ssu72 to distinguish among the different SP motifs. The phosphorylation of Tyr 1 further prohibited Ssu72 binding to pSer 2 and thereby prevented untimely Ser 2 dephosphorylation. Our results reveal critical roles for Tyr 1 in differentiating the phosphorylation states of Ser 2 /Ser 5 of CTD in RNA polymerase II that occur at different stages of transcription.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2021

Ser 71 Phosphorylation Inhibits Actin-Binding of Profilin-1 and Its Apoptosis-Sensitizing Activity

The essential actin-binding factor profilin-1 (Pfn1) is a non-classical tumor suppressor with the abilities toboth inhibit cellular proliferation and augment chemotherapy-induced apoptosis. Besides actin, Pfn1 interacts with proteins harboring the poly-L-proline (PLP) motifs. Our recent work demonstrated that both nuclear localization and PLP-binding are required for tumor growth inhibition by Pfn1, and this is at least partially due to Pfn1 association with the PLP-containing ENL protein in the Super Elongation Complex (SEC) and the transcriptional inhibition of pro-cancer genes. In this paper, by identifying a phosphorylation event of Pfn1 at Ser 71 capable of inhibiting its actin-binding and nuclear export, we provide in vitro and in vivo evidence that chemotherapy-induced apoptotic sensitization by Pfn1 requires its cytoplasmic localization and actin-binding. With regard to tumor growth inhibition byPfn1, our data indicate a requirement for dynamic actin association and dissociation rendered by reversible Ser 71 phosphorylation and dephosphorylation. Furthermore, genetic and pharmacological experiments showed that Ser 71 of Pfn1 can be phosphorylated by protein kinase A (PKA). Taken together, our data provide novel mechanistic insights into the multifaceted anticancer activities of Pfn1 and how they are spatially-defined in the cell and differentially regulated by ligand-binding.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Structures of HIV-1 Neutralizing Antibody 10E8 Delineate the Mechanistic Basis of Its Multi-Peak Behavior on Size-Exclusion Chromatography

Antibody 10E8 is capable of effectively neutralizing HIV through its recognition of the membrane-proximal external region (MPER), and a suitably optimized version of 10E8 might have utility in HIV therapy and prophylaxis. However, 10E8 displays a three-peak profile on size-exclusion chromatography (SEC), complicating its manufacture. Here we show cis-trans conformational isomerization of the Tyr-Pro-Pro (YPP) motif in the heavy chain 3rd complementarity-determining region (CDR H3) of antibody 10E8 to be the mechanistic basis of its multipeak behavior. We observed 10E8 to undergo slow conformational isomerization and delineate a mechanistic explanation for effective comodifiers that were able to resolve its SEC heterogeneity and to allow an evaluation of the critical quality attribute of aggregation. We determined crystal structures of single and double alanine mutants of a key di-proline motif and of a light chain variant, revealing alternative conformations of the CDR H3. We also replicated both multi-peak and delayed SEC behavior with MPER-antibodies 4E10 and VRC42, by introducing a Tyr-Pro (YP) motif into their CDR H3s. Our results show how a conformationally dynamic CDR H3 can provide the requisite structural plasticity needed for a highly hydrophobic paratope to recognize its membrane-proximal epitope.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Computational modeling highlights the role of the disordered Formin Homology 1 domain in profilin-actin transfer

Formins accelerate actin polymerization, assumed to occur through flexible Formin Homology 1 (FH1) domain-mediated transfer of profilin-actin to the barbed end. To investigate FH1 properties and address sequence effects, including varying length/distribution of profilin-binding proline-rich motifs, we performed all-atom simulations of a set of representative FH1 domains of formins: mouse mDia1 and mDia2, budding yeast Bni1 and Bnr1, and fission yeast Cdc12, For3, and Fus1. We discover that FH1 has flexible regions between high-propensity polyproline helix regions. A coarse-grained model retaining sequence specificity, assuming rigid polyproline segments, describes their size. Multiple bound profilins or profilin-actin complexes expand mDia1-FH1, which may be important in cells. Simulations of the barbed end bound to Bni1-FH1-FH2 dimer show that the leading FH1 can better transfer profilin or profilin-actin, with decreasing probability as the distance from FH2 increases.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Halophytes and heavy metals: A multi‐omics approach to understand the role of gene and genome duplication in the abiotic stress tolerance of Cakile maritima

Abstract Premise The origin of diversity is a fundamental biological question. Gene duplications are one mechanism that provides raw material for the emergence of novel traits, but evolutionary outcomes depend on which genes are retained and how they become functionalized. Yet, following different duplication types (polyploidy and tandem duplication), the events driving gene retention and functionalization remain poorly understood. Here we usedCakile maritima, a species that is tolerant to salt and heavy metals and shares an ancient whole‐genome triplication with closely related salt‐sensitive mustard crops (Brassica), as a model to explore the evolution of abiotic stress tolerance following polyploidy. Methods Using a combination of ionomics, free amino acid profiling, and comparative genomics, we characterize aspects of salt stress response inC. maritimaand identify retained duplicate genes that have likely enabled adaptation to salt and mild levels of cadmium. Results Cakile maritimais tolerant to both cadmium and salt treatments through uptake of cadmium in the roots. Proline constitutes greater than 30% of the free amino acid pool inC. maritimaand likely contributes to abiotic stress tolerance. We find duplicated gene families are enriched in metabolic and transport processes and identify key transport genes that may be involved inC. maritimaabiotic stress tolerance. Conclusions These findings identify pathways and genes that could be used to enhance plant resilience and provide a putative understanding of the roles of duplication types and retention on the evolution of abiotic stress response.

Plant Sciences↗

DOE OSTI2018

Dietary nutrient levels alter the metabolism of arginine family amino acids in the conceptus of Huanjiang mini‐pigs

Abstract BACKGROUND The arginine family amino acids (AFAAs) exert important roles in the metabolism, growth and development of the conceptus. However, to date, few studies have investigated the effects of maternal nutrient levels on the concentrations and metabolism of AFAAs in the conceptus. RESULTS Compared to low nutrient diets, high nutrient diets increased ( P < 0.05) the concentrations of citrulline and proline (Pro) in plasma; the concentrations of arginine, glutamine, Pro and ornithine (Orn) in the amniotic fluid; and the concentrations of all detected AFAAs in the allantoic fluid, which were most pronounced on day 45 of pregnancy. High nutrient diets upregulated ( P < 0.05) mRNA expression of arginase I ( Arg I ), Pro oxidase and spermidine synthetase ( SRM ) in the fetal placenta, as well as Arg II , SRM and spermine synthetase ( SMS ) expression in the fetal liver (most pronounced on day 45 of pregnancy). The same effect was observed for mRNA expression of NO synthase and Orn aminotransferase ( OAT ), mainly on day 110 of pregnancy, and for mRNA expression of Arg I , Arg II , OAT , Orn decarboxylase and SMS throughout pregnancy. High nutrient diets upregulated ( P < 0.05) mRNA expression of Y + L‐type amino acid transporter ( LAT ) and cationic amino acid transporter 1 ( CAT1 ) in the fetal jejunum throughout pregnancy. Dietary treatments did not affect ( P > 0.05) mRNA expression of Y + LAT1 , sodium‐coupled neutral amino acid transporter 2 ( SNAT2 ) and CAT1 in the fetal placenta, skeletal muscle and colon. CONCLUSION High nutrient diets increased the concentration and transport of AFAAs in the mothers and conceptus, which likely improves growth and development of the conceptus. © 2018 Society of Chemical Industry

Duan, Yehui↗

DOE OSTI2023

Specificity determinants revealed by the structure of glycosyltransferase Campylobacter concisus PglA

Abstract In selected Campylobacter species, the biosynthesis of N‐linked glycoconjugates via the pgl pathway is essential for pathogenicity and survival. However, most of the membrane‐associated GT‐B fold glycosyltransferases responsible for diversifying glycans in this pathway have not been structurally characterized which hinders the understanding of the structural factors that govern substrate specificity and prediction of resulting glycan composition. Herein, we report the 1.8 Å resolution structure of Campylobacter concisus PglA, the glycosyltransferase responsible for the transfer of N ‐acetylgalatosamine (GalNAc) from uridine 5′‐diphospho‐ N ‐acetylgalactosamine (UDP‐GalNAc) to undecaprenyl‐diphospho‐ N , N ′‐diacetylbacillosamine (UndPP‐diNAcBac) in complex with the sugar donor GalNAc. This study identifies distinguishing characteristics that set PglA apart within the GT4 enzyme family. Computational docking of the structure in the membrane in comparison to homologs points to differences in interactions with the membrane‐embedded acceptor and the structural analysis of the complex together with bioinformatics and site‐directed mutagenesis identifies donor sugar binding motifs. Notably, E113, conserved solely among PglA enzymes, forms a hydrogen bond with the GalNAc C6″‐OH. Mutagenesis of E113 reveals activity consistent with this role in substrate binding, rather than stabilization of the oxocarbenium ion transition state, a function sometimes ascribed to the corresponding residue in GT4 homologs. The bioinformatic analyses reveal a substrate‐specificity motif, showing that Pro281 in a substrate binding loop of PglA directs configurational preference for GalNAc over GlcNAc. This proline is replaced by a conformationally flexible glycine, even in distant homologs, which favor substrates with the same stereochemistry at C4, such as glucose. The signature loop is conserved across all Campylobacter PglA enzymes, emphasizing its importance in substrate specificity.

Vuksanovic, Nemanja↗

DOE OSTI2019

Substance P in the Gas Phase: Conformational Changes and Dissociations Induced by Collisional Activation in a Drift Tube

The work presented below is related to our companion paper in this issue, entitled: Substance P in solution: trans-to-cis configurational changes of penultimate prolines initiate non-enzymatic peptide bond cleavages. Two-dimensional ion mobility spectrometry (IMS-IMS) and mass spectrometry techniques are used to investigate structural transitions for [M+3H]{sup 3+} ions of substance P (subP) upon collisional activation (CA) in the gas phase. In this approach, different conformations of ions having a specified mobility are selected after an initial IMS separation, collisionally activated to produce new conformers, and these product structures are separated again using a second IMS region. In this way, it is possible to follow folding and unfolding transitions of different conformations. The analysis shows evidence for five conformations. Unlike other systems, every transition is irreversible. Studies as a function of activation voltage are used to discern pathways of structural changes prior to reaching the energy required for dissociation. Thresholds associated with the onsets of transitions are calibrated to obtain estimates of the energetic barriers between different structures and semi-quantitative potential energy diagrams are presented. Overall, barriers associated with structural transitions of [subP+3H]{sup 3+} in the absence of solvent are on the order of ~ 40 kJ mol{sup −1}, substantially lower than the ~ 90 kJ mol{sup −1} required for some similar structural transitions in solutions of ethanol. Comparisons of the transition energies in the gas phase with thermochemistry for similar transitions in solution provide clues about why reverse transitions are prohibited. .

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗

DOE OSTI2021

Towards understanding the formation of internal fragments generated by collisionally activated dissociation for top-down mass spectrometry

Top-down mass spectrometry (TD-MS) generates fragment ions that returns information on the polypeptide amino acid sequence. In addition to terminal fragments, internal fragments that result from multiple cleavage events can also be formed. Traditionally, internal fragments are largely ignored due to a lack of available software to reliably assign them, mainly caused by a poor understanding of their formation mechanism. To accurately assign internal fragments, their formation process needs to be better understood. Here we applied a statistical method to compare fragmentation patterns of internal and terminal fragments of peptides and proteins generated by collisionally activated dissociation (CAD). Internal fragments share similar fragmentation propensities with terminal fragments (e.g., enhanced cleavages N-terminal to proline and C-terminal to acidic residues), suggesting that their formation follows conventional CAD pathways. Internal fragments should be generated by subsequent cleavages of terminal fragments and their formation can be explained by the well-known mobile proton model. Additionally, internal fragments can be coupled with terminal fragments to form complementary product ions that span the entire protein sequence. These enhance our understanding of internal fragment formation and can help improve sequencing algorithms to accurately assign internal fragments, which will ultimately lead to more efficient and comprehensive TD-MS analysis of proteins and proteoforms.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2022

Multi-omics profiling of the cold tolerant Monoraphidium minutum 26B-AM in response to abiotic stress

Microalgae that are of interest for biofuel production must be able to tolerate environmental changes that occur in outdoor cultivation systems. While algal cultures may experience daily temperature fluctuations and seasonal environmental changes, the underlying mechanisms that control and regulate physiological responses and adaptation to environmental pressures are largely unknown. Systems-level characterization enabled by functional genomics can help identify biochemical pathways that promote stability and productivity of algae in various environmental conditions. Monoraphidium minutum 26B-AM, a freshwater green microalga, was identified as a top performer in biomass production in winter season screens. We sequenced the genome of M. minutum 26B-AM and applied our multi-omics pipeline to profile this high potential strain under high salt and cold temperature perturbations. Through comparative analysis, including other green algae in the class Chlorophyceae, we identified gene families unique to the genus Monoraphidium, including a desaturase that has been linked to cold tolerance in plants. We observed that osmolytes, such as trehalose, proline and betaine, accumulate under salt stress, coinciding with upregulation of genes involved in biosynthesis of these metabolites. From the genome annotation, we reconstructed a metabolic model to provide a detailed map of the metabolic pathways and can be used to simulate growth and reaction fluxes. This multi-omics analysis provides a foundation to explore algal strain potential for biofuel applications, guides strain engineering, and expands our understanding of metabolic and regulatory mechanisms of algae in applied systems.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2017

Functional characterisation of parvulin-type peptidyl prolyl cis-trans isomerase, PinA in Dictyostelium discoideum

Pin1-type parvulins are unique among PPIases that can catalyse an otherwise slow cis-trans isomerisation of phosphorylated peptide bond preceding proline in target proteins. This prolyl isomerisation process can regulate activity, stability and localisation of target proteins and thus control cellular processes like eukaryotic cell proliferation, cell cycle progression and gene regulation. Towards understanding the function of Pin1-type prolyl isomerisation in Dictyostelium discoideum, a slime mould with distinct growth and developmental phases, we identified PinA as a novel Pin1-type parvulin by its ability to complement the temperature sensitivity phenotype associated with a mutation in ESS1 in S. cerevisiae. In D. discoideum, pinA is temporally and spatially regulated during growth and development. PinA is both nuclear as well as cytoplasmic in the growing cells. We further show that loss of pinA (pinA{sup −}) leads to decreased growth rate, reduced spore formation and abnormal prespore-prestalk patterning. We conclude that PinA is required for normal growth as well as development in D. discoideum. - Highlights: • PinA is a bona fide homologue of S. cerevisiae Ess1. • PinA is required for normal cell proliferation of D. discoideum. • PinA is spatially localised in developmental structures. • PinA is important for cell differentiation and patterning.

60 APPLIED LIFE SCIENCES↗