570 Biowissenschaften; Biologie
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Bacterial molybdoenzymes are key enzymes involved in the global sulphur, nitrogen and carbon cycles. These enzymes require the insertion of the molybdenum cofactor (Moco) into their active sites and are able to catalyse a large range of redox-reactions. Escherichia coli harbours nineteen different molybdoenzymes that require a tight regulation of their synthesis according to substrate availability, oxygen availability and the cellular concentration of molybdenum and iron. The synthesis and assembly of active molybdoenzymes are regulated at the level of transcription of the structural genes and of translation in addition to the genes involved in Moco biosynthesis. The action of global transcriptional regulators like FNR, NarXL/QP, Fur and ArcA and their roles on the expression of these genes is described in detail. In this review we focus on what is known about the molybdenum- and iron-dependent regulation of molybdoenzyme and Moco biosynthesis genes in the model organism E. coli. The gene regulation in E. coli is compared to two other well studied model organisms Rhodobacter capsulatus and Shewanella oneidensis.
Molybdenum cofactor (Moco) biosynthesis is a complex process that involves the coordinated function of several proteins. In recent years it has become obvious that the availability of iron plays an important role in the biosynthesis of Moco. First, the MoaA protein binds two (4Fe-4S] clusters per monomer. Second, the expression of the moaABCDE and moeAB operons is regulated by FNR, which senses the availability of oxygen via a functional NFe-4S) cluster. Finally, the conversion of cyclic pyranopterin monophosphate to molybdopterin requires the availability of the L-cysteine desulfurase IscS, which is a shared protein with a main role in the assembly of Fe-S clusters. In this report, we investigated the transcriptional regulation of the moaABCDE operon by focusing on its dependence on cellular iron availability. While the abundance of selected molybdoenzymes is largely decreased under iron-limiting conditions, our data show that the regulation of the moaABCDE operon at the level of transcription is only marginally influenced by the availability of iron. Nevertheless, intracellular levels of Moco were decreased under iron-limiting conditions, likely based on an inactive MoaA protein in addition to lower levels of the L-cysteine desulfurase IscS, which simultaneously reduces the sulfur availability for Moco production. IMPORTANCE FNR is a very important transcriptional factor that represents the master switch for the expression of target genes in response to anaerobiosis. Among the FNR-regulated operons in Escherichia coli is the moaABCDE operon, involved in Moco biosynthesis. Molybdoenzymes have essential roles in eukaryotic and prokaryotic organisms. In bacteria, molybdoenzymes are crucial for anaerobic respiration using alternative electron acceptors. This work investigates the connection of iron availability to the biosynthesis of Moco and the production of active molybdoenzymes.
Larix populations at the tundra-taiga ecotone in northern Siberia are highly under-represented in population genetic studies, possibly due to the remoteness of these regions that can only be accessed at extraordinary expense. The genetic signatures of populations in these boundary regions are therefore largely unknown. We aim to generate organelle reference genomes for the detection of single nucleotide polymorphisms (SNPs) that can be used for paleogenetic studies. We present 19 complete chloroplast genomes and mitochondrial genomic sequences of larches from the southern lowlands of the Taymyr Peninsula (northernmost range of Larix gmelinii (Rupr.) Kuzen.), the lower Omoloy River, and the lower Kolyma River (both in the range of Larix cajanderi Mayr). The genomic data reveal 84 chloroplast SNPs and 213 putatively mitochondrial SNPs. Parsimony-based chloroplast haplotype networks show no spatial structure of individuals from different geographic origins, while the mitochondrial haplotype network shows at least a slight spatial structure with haplotypes from the Omoloy and Kolyma populations being more closely related to each other than to most of the haplotypes from the Taymyr populations. Whole genome alignments with publicly available complete chloroplast genomes of different Larix species show that among official plant barcodes only the rcbL gene contains sufficient polymorphisms, but has to be sequenced completely to distinguish the different provenances. We provide 8 novel mitochondrial SNPs that are putatively diagnostic for the separation of L. gmelinii and L. cajanderi, while 4 chloroplast SNPs have the potential to distinguish the L. gmelinii/ L. cajanderi group from other Larix species. Our organelle references can be used for a targeted primer and probe design allowing the generation of short amplicons. This is particularly important with regard to future investigations of, for example, the biogeographic history of Larix by screening ancient sedimentary DNA of Larix.
RNA-based processes play key roles in the regulation of eukaryotic gene expression. This includes both the processing of pre-mRNAs into mature mRNAs ready for translation and RNA-based silencing processes, such as RNA-directed DNA methylation (RdDM). Polyadenylation of pre-mRNAs is one important step in their processing and is carried out by three functionally specialized canonical nuclear poly(A) polymerases in Arabidopsis thaliana. Null mutations in one of these, termed PAPS1, result in a male gametophytic defect. Using a fluorescence-labelling strategy, we have characterized this defect in more detail using RNA and small-RNA sequencing. In addition to global defects in the expression of pollen-differentiation genes, paps1 null-mutant pollen shows a strong overaccumulation of transposable element (TE) transcripts, yet a depletion of 21- and particularly 24-nucleotide-long short interfering RNAs (siRNAs) and microRNAs (miRNAs) targeting the corresponding TEs. Double-mutant analyses support a specific functional interaction between PAPS1 and components of the RdDM pathway, as evident from strong synergistic phenotypes in mutant combinations involving paps1, but not paps2 paps4, mutations. In particular, the double-mutant of paps1 and rna-dependent rna polymerase 6 (rdr6) shows a synergistic developmental phenotype disrupting the formation of the transmitting tract in the female gynoecium. Thus, our findings in A. thaliana uncover a potentially general link between canonical poly(A) polymerases as components of mRNA processing and RdDM, reflecting an analogous interaction in fission yeast.
Electrosynthesis and characterization of molecularly imprinted polymers for peptides and proteins
(2019)
Recent investigations propose the acid sphingomyelinase (ASM)/ceramide system as a novel target for antidepressant action. ASM catalyzes the breakdown of the abundant membrane lipid sphingomyelin to the lipid messenger ceramide. This ASM‐induced lipid modification induces a local shift in membrane properties, which influences receptor clustering and downstream signaling. Canonical transient receptor potential channels 6 (TRPC6) are non‐selective cation channels located in the cell membrane that play an important role in dendritic growth, synaptic plasticity and cognition in the brain. They can be activated by hyperforin, an ingredient of the herbal remedy St. John’s wort for treatment of depression disorders. Because of their role in the context of major depression, we investigated the crosstalk between the ASM/ceramide system and TRPC6 ion channels in a pheochromocytoma cell line 12 neuronal cell model (PC12 rat pheochromocytoma cell line). Ca2+ imaging experiments indicated that hyperforin‐induced Ca2+ influx through TRPC6 channels is modulated by ASM activity. While antidepressants, known as functional inhibitors of ASM activity, reduced TRPC6‐mediated Ca2+ influx, extracellular application of bacterial sphingomyelinase rebalanced TRPC6 activity in a concentration‐related way. This effect was confirmed in whole‐cell patch clamp electrophysiology recordings. Lipidomic analyses revealed a decrease in very long chain ceramide/sphingomyelin molar ratio after ASM inhibition, which was connected with changes in the abundance of TRPC6 channels in flotillin‐1–positive lipid rafts as visualized by western blotting. Our data provide evidence that the ASM/ceramide system regulates TRPC6 channels likely by controlling their recruitment to specific lipid subdomains and thereby fine‐tuning their physical properties.
Cotton (Gossypium hirsutum) fibres consist of single cells that grow in a highly polarized manner, assumed to be controlled by the cytoskeleton(1-3). However, how the cytoskeletal organization and dynamics underpin fibre development remains unexplored. Moreover, it is unclear whether cotton fibres expand via tip growth or diffuse growth(2-4). We generated stable transgenic cotton plants expressing fluorescent markers of the actin and microtubule cytoskeleton. Live-cell imaging revealed that elongating cotton fibres assemble a cortical filamentous actin network that extends along the cell axis to finally form actin strands with closed loops in the tapered fibre tip. Analyses of F-actin network properties indicate that cotton fibres have a unique actin organization that blends features of both diffuse and tip growth modes. Interestingly, typical actin organization and endosomal vesicle aggregation found in tip-growing cell apices were not observed in fibre tips. Instead, endomembrane compartments were evenly distributed along the elongating fibre cells and moved bi-directionally along the fibre shank to the fibre tip. Moreover, plus-end tracked microtubules transversely encircled elongating fibre shanks, reminiscent of diffusely growing cells. Collectively, our findings indicate that cotton fibres elongate via a unique tip-biased diffuse growth mode.
In plants, transcripts move to distant body parts to potentially act as systemic signals regulating development and growth. Thousands of messenger RNAs (mRNAs) are transported across graft junctions via the phloem to distinct plant parts. Little is known regarding features, structural motifs, and potential base modifications of transported transcripts and how these may affect their mobility. We identified Arabidopsis thalianam RNAs harboring the modified base 5-methylcytosine (m(5)C) and found that these are significantly enriched in mRNAs previously described as mobile, moving over graft junctions to distinct plant parts. We confirm this finding with graft-mobile methylated mRNAs TRANSLATIONALLY CONTROLLED TUMOR PROTEIN 1 (TCTP1) and HEAT SHOCK COGNATE PROTEIN 70.1 (HSC70.1), whose mRNA transport is diminished in mutants deficient in m(5)C mRNA methylation. Together, our results point toward an essential role of cytosine methylation in systemic mRNA mobility in plants and that TCTP1 mRNA mobility is required for its signaling function.
Enhancers are critical for developmental stage-specific gene expression, but their dynamic regulation in plants remains poorly understood. Here we compare genome-wide localization of H3K27ac, chromatin accessibility and transcriptomic changes during flower development in Arabidopsis. H3K27ac prevalently marks promoter-proximal regions, suggesting that H3K27ac is not a hallmark for enhancers in Arabidopsis. We provide computational and experimental evidence to confirm that distal DNase. hypersensitive sites are predictive of enhancers. The predicted enhancers are highly stage-specific across flower development, significantly associated with SNPs for flowering-related phenotypes, and conserved across crucifer species. Through the integration of genome-wide transcription factor (TF) binding datasets, we find that floral master regulators and stage-specific TFs are largely enriched at developmentally dynamic enhancers. Finally, we show that enhancer clusters and intronic enhancers significantly associate with stage-specific gene regulation by floral master TFs. Our study provides insights into the functional flexibility of enhancers during plant development, as well as hints to annotate plant enhancers.
Inducible defences against predation are widespread in the natural world, allowing prey to economise on the costs of defence when predation risk varies over time or is spatially structured. Through interspecific interactions, inducible defences have major impacts on ecological dynamics, particularly predator-prey stability and phase lag. Researchers have developed multiple distinct approaches, each reflecting assumptions appropriate for particular ecological communities. Yet, the impact of inducible defences on ecological dynamics can be highly sensitive to the modelling approach used, making the choice of model a critical decision that affects interpretation of the dynamical consequences of inducible defences. Here, we review three existing approaches to modelling inducible defences: Switching Function, Fitness Gradient and Optimal Trait. We assess when and how the dynamical outcomes of these approaches differ from each other, from classic predator-prey dynamics and from commonly observed eco-evolutionary dynamics with evolving, but non-inducible, prey defences. We point out that the Switching Function models tend to stabilise population dynamics, and the Fitness Gradient models should be carefully used, as the difference with evolutionary dynamics is important. We discuss advantages of each approach for applications to ecological systems with particular features, with the goal of providing guidelines for future researchers to build on.
In nature, plants interact with numerous beneficial or pathogenic soil-borne microorganisms. Plants have developed various defense strategies to expel pathogenic microbes, some of which function soon after pathogen infection. We used Medicago truncatula and its oomycete pathogen Aphanomyces euteiches to elucidate early responses of the infected root. A. euteiches causes root rot disease in legumes and is a limiting factor in legume production. Transcript profiling of seedlings and adult plant roots inoculated with A. euteiches zoospores for 2 h revealed specific upregulation of a gene encoding a putative sesquiterpene synthase (M. truncatula TERPENE SYNTHASE 10 [MtTPS10]) in both developmental stages. MtTPS10 was specifically expressed in roots upon oomycete infection. Heterologous expression of MtTPS10 in yeast led to production of a blend of sesquiterpenes and sesquiterpene alcohols, with NMR identifying a major peak corresponding to himalachol. Moreover, plants carrying a tobacco (Nicotiana tabacum) retrotransposon Tnt1 insertion in MtTPS10 lacked the emission of sesquiterpenes upon A. euteiches infection, supporting the assumption that the identified gene encodes a multiproduct sesquiterpene synthase. Mttps10 plants and plants with reduced MtTPS10 transcript levels created by expression of an MtTPS10-artificial microRNA in roots were more susceptible to A. euteiches infection than were the corresponding wild-type plants and roots transformed with the empty vector, respectively. Sesquiterpenes produced by expression of MtTPS10 in yeast also inhibited mycelial growth and A. euteiches zoospore germination. These data suggest that sesquiterpene production in roots by MtTPS10 plays a previously unrecognized role in the defense response of M. truncatula against A. euteiches.
Identification and Characterization of Three Epithiospecifier Protein Isoforms in Brassica oleracea
(2019)
Glucosinolates present in Brassicaceae play a major role in herbivory defense. Upon tissue disruption, glucosinolates come into contact with myrosinase, which initiates their breakdown to biologically active compounds. Among these, the formation of epithionitriles is triggered by the presence of epithiospecifier protein (ESP) and a terminal double bond in the glucosinolate side chain. One ESP gene is characterized in the model plant Arabidopsis thaliana (AtESP; At1g54040.2). However, Brassica species underwent genome triplication since their divergence from the Arabidopsis lineage. This indicates the presence of multiple ESP isoforms in Brassica crops that are currently poorly characterized. We identified three B. oleracea ESPs, specifically BoESP1 (LOC106296341), BoESP2 (LOC106306810), and BoESP3 (LOC106325105) based on in silico genome analysis. Transcript and protein abundance were assessed in shoots and roots of four B. oleracea vegetables, namely broccoli, kohlrabi, white, and red cabbage, because these genotypes showed a differential pattern for the formation of glucosinolate hydrolysis products as well for their ESP activity. BoESP1 and BoESP2 were expressed mainly in shoots, while BoESP3 was abundant in roots. Biochemical characterization of heterologous expressed BoESP isoforms revealed different substrate specificities towards seven glucosinolates: all isoforms showed epithiospecifier activity on alkenyl glucosinolates, but not on non-alkenyl glucosinolates. The pH-value differently affected BoESP activity: while BoESP1 and BoESP2 activities were optimal at pH 6-7, BoESP3 activity remained relatively stable from pH 4 to 7. In order test their potential for the in vivo modification of glucosinolate breakdown, the three isoforms were expressed in A. thaliana Hi-0, which lacks AtESP expression, and analyzed for the effect on their respective hydrolysis products. The BoESPs altered the hydrolysis of allyl glucosinolate in the A. thaliana transformants to release 1-cyano-2,3-epithiopropane and reduced formation of the corresponding 3-butenenitrile and allyl isothiocyanate. Plants expressing BoESP2 showed the highest percentage of released epithionitriles. Given these results, we propose a model for isoform-specific roles of B. oleracea ESPs in glucosinolate breakdown.
Aims/hypothesis This study aimed to evaluate associations of height as well as components of height (sitting height and leg length) with risk of type 2 diabetes and to explore to what extent associations are explainable by liver fat and cardiometabolic risk markers. Methods A case-cohort study within the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam study comprising 26,437 participants who provided blood samples was designed. We randomly selected a subcohort of 2500 individuals (2029 diabetes-free at baseline and with anamnestic, anthropometrical and metabolic data for analysis). Of the 820 incident diabetes cases identified in the full cohort during 7 years of follow-up, 698 remained for analyses after similar exclusions. Results After adjustment for age, potential lifestyle confounders, education and waist circumference, greater height was related to lower diabetes risk (HR per 10 cm, men 0.59 [95% CI 0.47, 0.75] and women 0.67 [0.51, 0.88], respectively). Leg length was related to lower risk among men and women, but only among men if adjusted for total height. Adjustment for liver fat and triacylglycerols, adiponectin and C-reactive protein substantially attenuated associations between height and diabetes risk, particularly among women. Conclusions/interpretation We observed inverse associations between height and risk of type 2 diabetes, which was largely related to leg length among men. The inverse associations may be partly driven by lower liver fat content and a more favourable cardiometabolic profile.
Sphingolipids are a class of lipids that share a sphingoid base backbone. They exert various effects in eukaryotes, ranging from structural roles in plasma membranes to cellular signaling. De novo sphingolipid synthesis takes place in the endoplasmic reticulum (ER), where the condensation of the activated C₁₆ fatty acid palmitoyl-CoA and the amino acid L-serine is catalyzed by serine palmitoyltransferase (SPT). The product, 3-ketosphinganine, is then converted into more complex sphingolipids by additional ER-bound enzymes, resulting in the formation of ceramides. Since sphingolipid homeostasis is crucial to numerous cellular functions, improved assessment of sphingolipid metabolism will be key to better understanding several human diseases. To date, no assay exists capable of monitoring de novo synthesis sphingolipid in its entirety. Here, we have established a cell-free assay utilizing rat liver microsomes containing all the enzymes necessary for bottom-up synthesis of ceramides. Following lipid extraction, we were able to track the different intermediates of the sphingolipid metabolism pathway, namely 3-ketosphinganine, sphinganine, dihydroceramide, and ceramide. This was achieved by chromatographic separation of sphingolipid metabolites followed by detection of their accurate mass and characteristic fragmentations through high-resolution mass spectrometry and tandem-mass spectrometry. We were able to distinguish, unequivocally, between de novo synthesized sphingolipids and intrinsic species, inevitably present in the microsome preparations, through the addition of stable isotope-labeled palmitate-d₃ and L-serine-d₃. To the best of our knowledge, this is the first demonstration of a method monitoring the entirety of ER-associated sphingolipid biosynthesis. Proof-of-concept data was provided by modulating the levels of supplied cofactors (e.g., NADPH) or the addition of specific enzyme inhibitors (e.g., fumonisin B₁). The presented microsomal assay may serve as a useful tool for monitoring alterations in sphingolipid de novo synthesis in cells or tissues. Additionally, our methodology may be used for metabolism studies of atypical substrates – naturally occurring or chemically tailored – as well as novel inhibitors of enzymes involved in sphingolipid de novo synthesis.
We previously showed that purified 1-methoxy-3-indolylmethyl (1-MIM) glucosinolate, a secondary plant metabolite in Brassica species, is mutagenic in various in vitro systems and forms DNA and protein adducts in mouse models. In the present study, we administered 1-MIM glucosinolate in a natural matrix to mice, by feeding a diet containing pak choi powder and extract. Groups of animals were killed after 1, 2, 4 and 8 days of pak choi diet, directly or, in the case of the 8-day treatment, after 0, 8 and 16 days of recovery with pak choi-free diet. DNA adducts [N-2-(1-MIM)-dG, N-6-(1-MIM)-dA] in six tissues, as well as protein adducts [tau N-(1-MIM)-His] in serum albumin (SA) and hemoglobin (Hb) were determined using UPLC-MS/MS with isotopically labeled internal standards. None of the samples from the 12 control animals under standard diet contained any 1-MIM adducts. All groups receiving pak choi diet showed DNA adducts in all six tissues (exception: lung of mice treated for a single day) as well as SA and Hb adducts. During the feeding period, all adduct levels continuously increased until day 8 (in the jejunum until day 4). During the 14-day recovery period, N-2-(1-MIM)-dG in liver, kidney, lung, jejunum, cecum and colon decreased to 52, 41, 59, 11, 7 and 2%, respectively, of the peak level. The time course of N-6-(1-MIM)-dA was similar. Immunohistochemical analyses indicated that cell turnover is a major mechanism of DNA adduct elimination in the intestine. In the same recovery period, protein adducts decreased more rapidly in SA than in Hb, to 0.7 and 37%, respectively, of the peak level, consistent with the differential turnover of these proteins. In conclusion, the pak choi diet lead to the formation of high levels of adducts in mice. Cell and protein turnover was a major mechanism of adduct elimination, at least in gut and blood.
Enzyme immobilization using nanomaterials offers new approaches to enhanced bioelectrochemical performance and is essential for the preparation of bioelectrodes with high reproducibility and low cost. In this report, we describe the development of new three-dimensional (3D) bioelectrodes by immobilizing a "bioink" of glucose oxidase (GOD) in a matrix of reduced graphene oxides (RGOs), polyethylenimine (PEI), and ferrocene carboxylic acid (FcCOOH) on carbon paper (CP). CP with 3D interwoven carbon fibers serves as a solid porous and electronically conducting skeleton, providing large surface areas and space for loading the bioink and diffusion of substrate molecules, respectively. RGO enhances contact between the GOD-matrix and CP, maintaining high conductivity. The composition of the bioink has been systematically optimized. The GOD bioelectrodes show linearly increasing electrocatalytic oxidation current toward glucose concentration up to 48 mM. A hybrid enzymatic biofuel cell equipped with the GOD bioelectrode as a bioanode and a platinum cathode furthermore registers a maximum power density of 5.1 mu W cm(-2) and an open circuit voltage of 0.40 V at 25 degrees C. The new method reported of preparing a bioelectrode by drop-casting the bioink onto the substrate electrode is facile and versatile, with the potential of application also for other enzymatic bioelectrodes.
Proteasomes are key proteases in regulating protein homeostasis. Their holo-enzymes are composed of 40 different subunits which are arranged in a proteolytic core (CP) flanked by one to two regulatory particles (RP). Proteasomal proteolysis is essential for the degradation of proteins which control time-sensitive processes like cell cycle progression and stress response. In dividing yeast and human cells, proteasomes are primarily nuclear suggesting that proteasomal proteolysis is mainly required in the nucleus during cell proliferation. In yeast, which have a closed mitosis, proteasomes are imported into the nucleus as immature precursors via the classical import pathway. During quiescence, the reversible absence of proliferation induced by nutrient depletion or growth factor deprivation, proteasomes move from the nucleus into the cytoplasm. In the cytoplasm of quiescent yeast, proteasomes are dissociated into CP and RP and stored in membrane-less cytoplasmic foci, named proteasome storage granules (PSGs). With the resumption of growth, PSGs clear and mature proteasomes are transported into the nucleus by Blm10, a conserved 240 kDa protein and proteasome-intrinsic import receptor. How proteasomes are exported from the nucleus into the cytoplasm is unknown.
Aims Plant-plant interactions, being positive or negative, are recognized to be key factors in structuring plant communities. However, it is thought that root competition may be less important than shoot competition due to greater size symmetry belowground. Because direct experimental tests on the importance of root competition are scarce, we aim at elucidating whether root competition may have direct or indirect effects on community structure. Indirect effects may occur by altering the overall size asymmetry of competition through root-shoot competitive interactions. Methods We used a phytometer approach to examine the effects of root, shoot and total competition intensity and importance on evenness of experimental plant communities. Thereby two different phytometer species, Festuca brevipila and Dianthus carthusianorum, were grown in small communities of six grassland species over three levels of light and water availability, interacting with neighbouring shoots, roots, both or not at all. Important Findings We found variation in community evenness to be best explained if root and shoot (but not total) competition were considered. However, the effects were species specific: in Dianthus communities increasing root competition increased plant community evenness, while in Festuca communities shoot competition was the driving force of this evenness response. Competition intensities were influenced by environmental conditions in Dianthus, but not in Festuca phytometer plants. While we found no evidence for root-shoot interactions for neither phytometer species root competition in Dianthus communities led to increased allocation to shoots, thereby increasing the potential ability to perform in size-asymmetric competition for light. Our experiment demonstrates the potential role of root competition in structuring plant communities.
Hormonal regulation of neuronal mitochondrial unfolded protein response and its impact on metabolism
(2019)
The hypothalamus is the main brain area of central regulation of whole body metabolism through impacting food intake and energy expenditure. For the complex regulation, high amounts of energy are needed and mainly provided by mitochondria. Hence, mitochondrial function is crucial for cell homeostasis and modulates central insulin sensitivity. Thus, mitochondrial dysfunction is associated with insulin resistance in the brain and therefore is involved in the pathogenesis of type-2 diabetes (T2D). Mitochondrial health and protein homeostasis is propagated by mitochondrial stress responses like e.g. mitochondrial unfolded protein response (UPRmt). Therefore, studies regarding the regulation of mitochondrial homeostasis are crucial for understanding its effects on the central nervous system (CNS) for the progression of metabolic and nutrition-dependent disorders.
One main aim of this thesis was to investigate the metabolic regulation of mitochondrial stress responsiveness in the hypothalamus. The observed results showed that functional ERK-dependent insulin signaling is needed for regulation of mitochondrial stress response (MSR) genes and positively impacted the metabolism by controlling mitochondrial proteostasis without affecting mitochondrial biogenesis.
To further explore the role of MSR genes for brain cell homeostasis and its consequences for the metabolism, one of the key players - the mitochondrial chaperone heat shock protein 10 (Hsp10) – was studied in detail. Hsp10 expression was decreased in insulin-resistant, hyperglycemic db/db mice brains along with increased protein oxidation. Leptin, another key hormone in regulating metabolism, was able to induce Hsp10 in neurons. Appropriately, lentiviral-mediated knock down (KD) of Hsp10 introduced into hypothalamic CLU-183 cells induced mitochondrial dysfunction, altered mitochondrial dynamics and increased contact sites between mitochondria and endoplasmic reticulum (ER). In addition, Hsp10 KD caused cellular insulin resistance along with increasing oxidative stress specifically in mitochondrial fraction.
Interestingly, acute Hsp10 KD in the arcuate nucleus of the hypothalamus in C57BL/6N male mice did not change body weight or food intake, but it increased plasma leptin concentrations suggesting an effect on global leptin signaling. It increased hepatic markers of gluconeogenesis and hepatic insulin resistance along with features of low-grade inflammation.
Long-term studies of hypothalamic Hsp10 KD mice revealed unaltered systemic insulin sensitivity. The demonstrated increase in markers of hepatic gluconeogenesis of acute Hsp10 KD was still exhibited after 13 weeks, but insulin resistance in the liver was no longer observed.
In conclusion, hypothalamic insulin action regulates MSR and ensures proper mitochondrial function which positively affects metabolism. In addition, hypothalamic Hsp10 acts as a modulator of both insulin and leptin signaling and is identified as pivotal for the regulation of central mitochondrial function as well as insulin sensitivity in the brain and it impacts liver function. It may present a regulator of brain-liver crosstalk influencing hepatic gluconeogenesis and insulin sensitivity through a novel regulatory signaling mechanism.
This is the eleventh of a series of miscellaneous contributions, by various authors, where hitherto unpublished data relevant to both the Med-Checklist and the Euro+Med (or Sisyphus) projects are presented. This instalment deals with the families Anacardiaceae, Asparagaceae (incl. Hyacinthaceae), Bignoniaceae, Cactaceae, Compositae, Cruciferae, Cyperaceae, Ericaceae, Gramineae, Labiatae, Leguminosae, Orobanchaceae, Polygonaceae, Rosaceae, Solanaceae and Staphyleaceae. It includes new country and area records and taxonomic and distributional considerations for taxa in Bidens, Campsis, Centaurea, Cyperus, Drymocallis, Engem, Hoffmannseggia, Hypopitys, Lavandula, Lithraea, Melilotus, Nicotiana, Olimarabidopsis, Opuntia, Orobanche, Phelipanche, Phragmites, Rumex, Salvia, Schinus, Staphylea, and a new combination in Drimia.
Malnutrition is widespread in older people and represents a major geriatric syndrome with multifactorial etiology and severe consequences for health outcomes and quality of life. The aim of the present paper is to describe current approaches and evidence regarding malnutrition treatment and to highlight relevant knowledge gaps that need to be addressed. Recently published guidelines of the European Society for Clinical Nutrition and Metabolism (ESPEN) provide a summary of the available evidence and highlight the wide range of different measures that can be taken—from the identification and elimination of potential causes to enteral and parenteral nutrition—depending on the patient’s abilities and needs. However, more than half of the recommendations therein are based on expert consensus because of a lack of evidence, and only three are concern patient-centred outcomes. Future research should further clarify the etiology of malnutrition and identify the most relevant causes in order to prevent malnutrition. Based on limited and partly conflicting evidence and the limitations of existing studies, it remains unclear which interventions are most effective in which patient groups, and if specific situations, diseases or etiologies of malnutrition require specific approaches. Patient-relevant outcomes such as functionality and quality of life need more attention, and research methodology should be harmonised to allow for the comparability of studies.
Intraspecific genetic diversity and distribution of North African hedgehogs (Mammalia: Erinaceidae)
(2019)
Despite growing efforts to halt biodiversity loss, knowledge of species diversity and distribution is highly geographically biased, leaving some areas unexplored. Taxa distributed in remote, desert areas, such as hedgehogs (Mammalia; Eulipotyphla) in North Africa, are good examples of current knowledge gaps in systematics and biogeography. Here we studied the geographical distribution and intraspecific genetic diversity of hedgehogs in North Africa. Specimens belonging to North African and Eurasian species were analysed with mitochondrial (control region, CR) and nuclear (recombination activating gene 1, RAG1) gene fragments. This revealed a broader geographical distribution of Atelerix algirus in south-western Libya and of Paraechinus aethiopicus along the Atlantic Sahara. High intraspecific genetic differentiation was found in A. algirus and A. albiventris at the mitochondrial level, with nuclear haplotype sharing across their ranges. These findings suggest that biogeographical patterns of hedgehogs in North Africa are more complex than previously suggested, highlighting a need for further investigation in this remote and poorly known area.
Sustained glacier melt in the Himalayas has gradually spawned more than 5,000 glacier lakes that are dammed by potentially unstable moraines. When such dams break, glacier lake outburst floods (GLOFs) can cause catastrophic societal and geomorphic impacts. We present a robust probabilistic estimate of average GLOFs return periods in the Himalayan region, drawing on 5.4 billion simulations. We find that the 100-y outburst flood has an average volume of 33.5(+3.7)/(-3.7) x 10(6) m(3) (posterior mean and 95% highest density interval [HDI]) with a peak discharge of 15,600(+2.000)/(-1,800) m(3).S-1. Our estimated GLOF hazard is tied to the rate of historic lake outbursts and the number of present lakes, which both are highest in the Eastern Himalayas. There, the estimated 100-y GLOF discharge (similar to 14,500 m(3).s(-1)) is more than 3 times that of the adjacent Nyainqentanglha Mountains, and at least an order of magnitude higher than in the Hindu Kush, Karakoram, and Western Himalayas. The GLOF hazard may increase in these regions that currently have large glaciers, but few lakes, if future projected ice loss generates more unstable moraine-dammed lakes than we recognize today. Flood peaks from GLOFs mostly attenuate within Himalayan headwaters, but can rival monsoon-fed discharges in major rivers hundreds to thousands of kilometers downstream. Projections of future hazard from meteorological floods need to account for the extreme runoffs during lake outbursts, given the increasing trends in population, infrastructure, and hydropower projects in Himalayan headwaters.
Bacterial pathogens are influenced by signaling molecules including the catecholamines adrenaline and noradrenaline which are host-derived hormones and neurotransmitters. Adrenaline and noradrenaline modulate growth, motility and virulence of bacteria. We show that adrenaline is converted by the pathogen Vibrio cholerae to adrenochrome in the course of respiration, and demonstrate that superoxide produced by the respiratory, Na+ - translocating NADH:quinone oxidoreductase (NQR) acts as electron acceptor in the oxidative conversion of adrenaline to adrenochrome. Adrenochrome stimulates growth of V. cholerae, and triggers specific responses in V. cholerae and in immune cells. We performed a quantitative proteome analysis of V. cholerae grown in minimal medium with glucose as carbon source without catecholamines, or with adrenaline, noradrenaline or adrenochrome. Significant regulation of proteins participating in iron transport and iron homeostasis, in energy metabolism, and in signaling was observed upon exposure to adrenaline, noradrenaline or adrenochrome. On the host side, adrenochrome inhibited lipopolysaccharide-triggered formation of TNF-alpha by THP-1 monocytes, though to a lesser extent than adrenaline. It is proposed that adrenochrome produced from adrenaline by respiring V. cholerae functions as effector molecule in pathogen-host interaction.
Determining the relationship between genotype and phenotype is the key to understand the plasticity and robustness of phenotypes in nature. While the directly observable plant phenotypes (e.g. agronomic, yield and stress resistance traits) have been well-investigated, there is still a lack in our knowledge about the genetic basis of intermediate phenotypes, such as metabolic phenotypes. Dissecting the links between genotype and phenotype depends on suitable statistical models. The state-of-the-art models are developed for directly observable phenotypes, regardless the characteristics of intermediate phenotypes. This thesis aims to fill the gaps in understanding genetic architecture of intermediate phenotypes, and how they tie to composite traits, namely plant growth. The metabolite levels and reaction fluxes, as two aspects of metabolic phenotypes, are shaped by the interrelated chemical reactions formed in genome-scale metabolic network. Here, I attempt to answer the question: Can the knowledge of underlying genome-scale metabolic network improve the model performance for prediction of metabolic phenotypes and associated plant growth? To this end, two projects are investigated in this thesis. Firstly, we propose an approach that couples genomic selection with genome-scale metabolic network and metabolic profiles in Arabidopsis thaliana to predict growth. This project is the first integration of genomic data with fluxes predicted based on constraint-based modeling framework and data on biomass composition. We demonstrate that our approach leads to a considerable increase of prediction accuracy in comparison to the state-of-the-art methods in both within and across environment predictions. Therefore, our work paves the way for combining knowledge on metabolic mechanisms in the statistical approach underlying genomic selection to increase the efficiency of future plant breeding approaches. Secondly, we investigate how reliable is genomic selection for metabolite levels, and which single nucleotide polymorphisms (SNPs), obtained from different neighborhoods of a given metabolic network, contribute most to the accuracy of prediction. The results show that the local structure of first and second neighborhoods are not sufficient for predicting the genetic basis of metabolite levels in Zea mays. Furthermore, we find that the enzymatic SNPs can capture most the genetic variance and the contribution of non-enzymatic SNPs is in fact small. To comprehensively understand the genetic architecture of metabolic phenotypes, I extend my study to a local Arabidopsis thaliana population and their hybrids. We analyze the genetic architecture in primary and secondary metabolism as well as in growth. In comparison to primary metabolites, compounds from secondary metabolism were more variable and show more non-additive inheritance patterns which could be attributed to epistasis. Therefore, our study demonstrates that heterozygosity in local Arabidopsis thaliana population generates metabolic variation and may impact several tasks directly linked to metabolism. The studies in this thesis improve the knowledge of genetic architecture of metabolic phenotypes in both inbreed and hybrid population. The approaches I proposed to integrate genome-scale metabolic network with genomic data provide the opportunity to obtain mechanistic insights about the determinants of agronomically important polygenic traits.
River ecosystems receive and process vast quantities of terrestrial organic carbon, the fate of which depends strongly on microbial activity. Variation in and controls of processing rates, however, are poorly characterized at the global scale. In response, we used a peer-sourced research network and a highly standardized carbon processing assay to conduct a global-scale field experiment in greater than 1000 river and riparian sites. We found that Earth’s biomes have distinct carbon processing signatures. Slow processing is evident across latitudes, whereas rapid rates are restricted to lower latitudes. Both the mean rate and variability decline with latitude, suggesting temperature constraints toward the poles and greater roles for other environmental drivers (e.g., nutrient loading) toward the equator. These results and data set the stage for unprecedented “next-generation biomonitoring” by establishing baselines to help quantify environmental impacts to the functioning of ecosystems at a global scale.
The great auk was once abundant and distributed across the North Atlantic. It is now extinct, having been heavily exploited for its eggs, meat, and feathers. We investigated the impact of human hunting on its demise by integrating genetic data, GPS-based ocean current data, and analyses of population viability. We sequenced complete mitochondrial genomes of 41 individuals from across the species' geographic range and reconstructed population structure and population dynamics throughout the Holocene. Taken together, our data do not provide any evidence that great auks were at risk of extinction prior to the onset of intensive human hunting in the early 16th century. In addition, our population viability analyses reveal that even if the great auk had not been under threat by environmental change, human hunting alone could have been sufficient to cause its extinction. Our results emphasise the vulnerability of even abundant and widespread species to intense and localised exploitation.
Polyhydroxyalkanoates (PHAs) have attracted attention as degradable (co)polyesters which can be produced by microorganisms with variations in the side chain. This structural variation influences not only the thermomechanical properties of the material but also its degradation behavior. Here, we used Langmuir monolayers at the air-water (A-W) interface as suitable models for evaluating the abiotic degradation of two PHAs with different side-chain lengths and crystallinity. By controlling the polymer state (semi crystalline, amorphous), the packing density, the pH, and the degradation mechanism, we could draw several significant conclusions. (i) The maximum degree of crystallinity for a PHA film to be efficiently degraded up to pH = 12.3 is 40%. (ii) PHA made of repeating units with shorter side-chain length are more easily hydrolyzed under alkaline conditions. The efficiency of alkaline hydrolysis decreased by about 65% when the polymer was 40% crystalline. (iii) In PHA films with a relatively high initial crystallinity, abiotic degradation initiated a chemicrystallization phenomenon, detected as an increase in the storage modulus (E'). This could translate into an increase in brittleness and reduction in the material degradability. Finally, we demonstrate the stability of the measurement system for long-term experiments, which allows degradation conditions for polymers that could closely simulate real-time degradation.
Marketing of adulterated beeswax foundation has recently become a major economic problem for beekeepers. Paraffin contamination leads to collapse of combs, and stearic acid has a negative influence on the development of bee brood. The quality of beeswax for beekeeping has not been standardized in EU regulations. Recently, it was shown that attenuated total reflectance Fourier-transform infrared spectroscopy (FTIR-ATR) can be used to determine beeswax adulteration. Differences in the IR spectra of authentic beeswax can be identified and calculated through comparison with authentic beeswax. In this study, the method is further validated by employing a high number of samples of authentic beeswax from different origins. Low quantification and detection limits are achieved for paraffin, stearic acid, tallow, carnauba wax, and candelilla wax. Furthermore, the FTIR-ATR analytical conditions are verified by analyzing 358 samples of commercial and beekeeper-produced beeswax foundations. Multi-adulterated samples with as many as five different additives in beeswax mixtures are identified with the same accuracy as single substances. Additionally, the spectra of a further 14 different natural and synthetic waxes and hardened fats are analyzed and are compared with beeswax. Finally, a spectral library is established that can be used for further studies. Practical Applications: FTIR-ATR is a fast and cost-efficient tool in beeswax analysis for accurately monitoring a high sample volume. Analysis of 358 beeswax foundations showed an adulteration of 21.8% of the samples with paraffin, stearic acid, tallow, and combinations. Based on the results of this study, it is possible to detect beeswax adulteration of less than 3% of these adulterants and their combinations by FTIR-ATR spectroscopy. This method can be used for monitoring beeswax foundations to identify adulterated materials, exclude these materials from the recycling process, and produce high-quality beeswax, which is essential for bee health.
The oligotrophic subtropical gyre covers a vast area of the Atlantic Ocean. Decades of time-series monitoring have generated detailed temporal information about zooplankton species and abundances at fixed locations within the gyre, but their live/dead status is often omitted, especially in the dynamic subtropical convergence zone (STCZ) where the water column stratification pattern can change considerably across the front as warm and cold water masses converge. We conducted a detailed survey in the North Atlantic STCZ and showed that over 85% of the copepods were typically concentrated in the upper 200 m. Copepod carcasses were present in all samples and their proportional numerical abundances increased with depth, reaching up to 91% at 300-400 m. Overall, 14-19% of the copepods within the upper 200 m were carcasses. Shipboard experiments showed that during carcass decomposition, microbial respiration increased, and the bacterial community associated with the carcasses diverged from that in the ambient water. Combining field and experimental data, we estimated that decomposing copepod carcasses constitute a negligible oxygen sink in the STCZ, but sinking carcasses may represent an overlooked portion of the passive carbon sinking flux and should be incorporated in future studies of carbon flux in this area.
We have developed a three-dimensional (3D) graphene electrode suitable for the immobilization of human sulfite oxidase (hSO), which catalyzes the electrochemical oxidation of sulfite via direct electron transfer (DET). The electrode is fabricated by drop-casting graphene-polyethylenimine (G-P) composites on carbon papers (CPs) precoated with graphene oxide (GO). The negatively charged hSO can be adsorbed electrostatically on the positively charged matrix (G-P) on CP electrodes coated with GO (CPG), with a proper orientation for accelerated DET. Notably, further electrochemical reduction of G-P on CPG electrodes leads to a 9-fold increase of the saturation catalytic current density (j(m)) for sulfite oxidation reaching 24.4 +/- 0.3 mu A to cm(-2), the highest value among reported DET-based hSO bioelectrodes. The increased electron transfer rate plays a dominating role in the enhancement of direct enzymatic current because of the improved electric contact of hSO with the electrode, The optimized hSO bioelectrode shows a significant catalytic rate (k(cat): 25.6 +/- 0.3 s(-1)) and efficiency (k(cat)/K-m: 0.231 +/- 0.003 s(-1) mu M-1) compared to the reported hSO bioelectrodes. The assembly of the hSO bioanode and a commercial platinum biocathode allows the construction of sulfite/O-2 enzymatic biofuel cells (EBFCs) with flowing fuels. The optimized EBFC displays an open-circuit voltage (OCV) of 0.64 +/- 0.01 V and a maximum power density of 61 +/- 6 mu W cm(-2) (122 +/- 12 mW m(-3)) at 30 degrees C, which exceeds the best reported value by more than 6 times.
Distinct cellular roles for PDCD10 define a gut-brain axis in cerebral cavernous malformation
(2019)
Cerebral cavernous malformation (CCM) is a genetic, cerebrovascular disease. Familial CCM is caused by genetic mutations in KRIT1, CCM2, or PDCD10. Disease onset is earlier and more severe in individuals with PDCD10 mutations. Recent studies have shown that lesions arise from excess mitogen-activated protein kinase kinase kinase 3 (MEKK3) signaling downstream of Toll-like receptor 4 (TLR4) stimulation by lipopolysaccharide derived from the gut microbiome. These findings suggest a gut-brain CCM disease axis but fail to define it or explain the poor prognosis of patients with PDCD10 mutations. Here, we demonstrate that the gut barrier is a primary determinant of CCM disease course, independent of microbiome configuration, that explains the increased severity of CCM disease associated with PDCD10 deficiency. Chemical disruption of the gut barrier with dextran sulfate sodium augments CCM formation in a mouse model, as does genetic loss of Pdcd10, but not Krit1, in gut epithelial cells. Loss of gut epithelial Pdcd10 results in disruption of the colonic mucosal barrier. Accordingly, loss of Mucin-2 or exposure to dietary emulsifiers that reduce the mucus barrier increases CCM burden analogous to loss of Pdcd10 in the gut epithelium. Last, we show that treatment with dexamethasone potently inhibits CCM formation in mice because of the combined effect of action at both brain endothelial cells and gut epithelial cells. These studies define a gut-brain disease axis in an experimental model of CCM in which a single gene is required for two critical components: gut epithelial function and brain endothelial signaling.
Persulfide groups participate in a wide array of biochemical pathways and are chemically very versatile. The TusA protein has been identified as a central element supplying and transferring sulfur as persulfide to a number of important biosynthetic pathways, like molybdenum cofactor biosynthesis or thiomodifications in nucleosides of tRNAs. In recent years, it has furthermore become obvious that this protein is indispensable for the oxidation of sulfur compounds in the cytoplasm. Phylogenetic analyses revealed that different TusA protein variants exists in certain organisms, that have evolved to pursue specific roles in cellular pathways. The specific TusA-like proteins thereby cannot replace each other in their specific roles and are rather specific to one sulfur transfer pathway or shared between two pathways. While certain bacteria like Escherichia coli contain several copies of TusA-like proteins, in other bacteria like Allochromatium vinosum a single copy of TusA is present with an essential role for this organism. Here, we give an overview on the multiple roles of the various TusA-like proteins in sulfur transfer pathways in different organisms to shed light on the remaining mysteries of this versatile protein.
Shrub encroachment has far-reaching ecological and economic consequences in many ecosystems worldwide. Yet, compositional changes associated with shrub encroachment are often overlooked despite having important effects on ecosystem functioning. We document the compositional change and potential drivers for a northern Namibian Combretum woodland transitioning into a Terminalia shrubland. We use a multiproxy record (pollen, sedimentary ancient DNA, biomarkers, compound-specific carbon (delta C-13) and deuterium (delta D) isotopes, bulk carbon isotopes (delta(13)Corg), grain size, geochemical properties) from Lake Otjikoto at high taxonomical and temporal resolution. We provide evidence that state changes in semiarid environments may occur on a scale of one century and that transitions between stable states can span around 80 years and are characterized by a unique vegetation composition. We demonstrate that the current grass/woody ratio is exceptional for the last 170 years, as supported by n-alkane distributions and the delta C-13 and delta(13)Corg records. Comparing vegetation records to environmental proxy data and census data, we infer a complex network of global and local drivers of vegetation change. While our delta D record suggests physiological adaptations of woody species to higher atmospheric pCO(2) concentration and drought, our vegetation records reflect the impact of broad-scale logging for the mining industry, and the macrocharcoal record suggests a decrease in fire activity associated with the intensification of farming. Impact of selective grazing is reflected by changes in abundance and taxonomical composition of grasses and by an increase of nonpalatable and trampling-resistant taxa. In addition, grain-size and spore records suggest changes in the erodibility of soils because of reduced grass cover. Synthesis. We conclude that transitions to an encroached savanna state are supported by gradual environmental changes induced by management strategies, which affected the resilience of savanna ecosystems. In addition, feedback mechanisms that reflect the interplay between management legacies and climate change maintain the encroached state.
Canavanine (CAN) is a nonproteinogenic amino acid synthesized in legumes. In mammalians, as arginine analogue, it is an inhibitor of nitric oxide synthase (NOS) activity. The aim of this study was to investigate the impact of CAN-induced nitric oxide level limitation on the antioxidant system and S-nitrosoglutathione (GSNO) metabolism in roots of tomato seedlings. Treatment with CAN (10 or 50 mu M) for 24-72 h led to restriction in root growth. Arginine-dependent NOS-like activity was almost completely inhibited, demonstrating direct effect of CAN action. CAN increased total antioxidant capacity and the level of sulphydryl groups. Catalase (CAT) and superoxide dismutase (SOD) activity decreased in CAN exposed roots. CAN supplementation resulted in the decrease of transcript levels of genes coding CAT (with the exception of CAT1). Genes coding SOD (except MnSOD and CuSOD) were upregulated by CAN short treatment; prolonged exposition to 50-mu M CAN resulted in downregulation of FeSOD, CuSOD, and SODP-2. Activity of glutathione reductase dropped down after short-term (10-mu M CAN) supplementation, while glutathione peroxidase activity was not affected. Transcript levels of glutathione reductase genes declined in response to CAN. Genes coding glutathione peroxidase were upregulated by 50-mu M CAN, while 10-mu M CAN downregulated GSHPx1. Inhibition of NOS-like activity by CAN resulted in lower GSNO accumulation in root tips. Activity of GSNO reductase was decreased by short-term supplementation with CAN. In contrast, GSNO reductase protein abundance was higher, while transcript levels were slightly altered in roots exposed to CAN. This is the first report on identification of differentially nitrated proteins in response to supplementation with nonproteinogenic amino acid. Among nitrated proteins differentially modified by CAN, seed storage proteins (after short-term CAN treatment) and components of the cellular redox system (after prolonged CAN supplementation) were identified. The findings demonstrate that due to inhibition of NOS-like activity, CAN leads to modification in antioxidant system. Limitation in GSNO level is due to lower nitric oxide formation, while GSNO catabolism is less affected. We demonstrated that monodehydroascorbate reductase, activity of which is inhibited in roots of CAN-treated plants, is the protein preferentially modified by tyrosine nitration.
Hantavirus assembly and budding are governed by the surface glycoproteins Gn and Gc. In this study, we investigated the glycoproteins of Puumala, the most abundant Hantavirus species in Europe, using fluorescently labeled wild-type constructs and cytoplasmic tail (CT) mutants. We analyzed their intracellular distribution, co-localization and oligomerization, applying comprehensive live, single-cell fluorescence techniques, including confocal microscopy, imaging flow cytometry, anisotropy imaging and Number&Brightness analysis. We demonstrate that Gc is significantly enriched in the Golgi apparatus in absence of other viral components, while Gn is mainly restricted to the endoplasmic reticulum (ER). Importantly, upon co-expression both glycoproteins were found in the Golgi apparatus. Furthermore, we show that an intact CT of Gc is necessary for efficient Golgi localization, while the CT of Gn influences protein stability. Finally, we found that Gn assembles into higher-order homo-oligomers, mainly dimers and tetramers, in the ER while Gc was present as mixture of monomers and dimers within the Golgi apparatus. Our findings suggest that PUUV Gc is the driving factor of the targeting of Gc and Gn to the Golgi region, while Gn possesses a significantly stronger self-association potential.
Cold-regulated (COR) 15A is an intrinsically disordered protein (IDP) from Arabidopsis thaliana important for freezing tolerance. During freezing-induced cellular dehydration, COR15A transitions from a disordered to mostly alpha-helical structure. We tested whether mutations that increase the helicity of COR15A also increase its protective function. Conserved glycine residues were identified and mutated to alanine. Nuclear magnetic resonance (NMR) spectroscopy was used to identify residue-specific changes in helicity for wildtype (WT) COR15A and the mutants. Circular dichroism (CD) spectroscopy was used to monitor the coil-helix transition in response to increasing concentrations of trifluoroethanol (TFE) and ethylene glycol. The impact of the COR15A mutants on the stability of model membranes during a freeze-thaw cycle was investigated by fluorescence spectroscopy. The results of these experiments showed the mutants had a higher content of alpha-helical structure and the increased alpha-helicity improved membrane stabilization during freezing. Comparison of the TFE- and ethylene glycol-induced coil-helix transitions support our conclusion that increasing the transient helicity of COR15A in aqueous solution increases its ability to stabilize membranes during freezing. Altogether, our results suggest the conserved glycine residues are important for maintaining the disordered structure of COR15A but are also compatible with the formation of alpha-helical structure during freezing induced dehydration.
Cold regulated protein 15A (COR15A) is a nuclear encoded, intrinsically disordered protein that is found in Arabidopsis thaliana. It belongs to the Late Embryogenesis Abundant (LEA) family of proteins and is responsible for increased freezing tolerance in plants. COR15A is intrinsically disordered in dilute solutions and adopts a helical structure upon dehydration or in the presence of co-solutes such as TFE and ethylene glycol. This helical structure is thought to be important for protecting plants from dehydration induced by freezing. Multiple protein sequence alignments revealed the presence of several conserved glycine residues that we hypothesize keeps COR15A from becoming helical in dilute solutions. Using AGADIR, the change in helical content of COR15A when these conserved glycine residues were mutated to alanine residues was predicted. Based on the predictions, glycine to alanine mutants were made at position 68, and 54,68,81, and 84. Labeled samples of wildtype COR15A and mutant proteins were purified and NMR experiments were performed to examine any structural changes induced by the mutations. To test the effects of dehydration on the structure of COR15A, trifluoroethanol, an alcohol based co solvent that is proposed to induce/stabilize helical structure in peptides was added to the NMR samples, and the results of the experiment showed an increase in helical content, compared to the samples without TFE. To test the functional differences between wild type and the mutants, liposome leakage assays were performed. The results from these assays suggest the more helical mutants may augment membrane stability.
Light and gravity are two key determinants in orientating plant stems for proper growth and development. The organization and dynamics of the actin cytoskeleton are essential for cell biology and critically regulated by actin-binding proteins. However, the role of actin cytoskeleton in shoot negative gravitropism remains controversial. In this work, we report that the actin-binding protein Rice Morphology Determinant (RMD) promotes reorganization of the actin cytoskeleton in rice (Oryza sativa) shoots. The changes in actin organization are associated with the ability of the rice shoots to respond to negative gravitropism. Here, light-grown rmd mutant shoots exhibited agravitropic phenotypes. By contrast, etiolated rmd shoots displayed normal negative shoot gravitropism. Furthermore, we show that RMD maintains an actin configuration that promotes statolith mobility in gravisensing endodermal cells, and for proper auxin distribution in light-grown, but not dark-grown, shoots. RMD gene expression is diurnally controlled and directly repressed by the phytochrome-interacting factor-like protein OsPIL16. Consequently, overexpression of OsPIL16 led to gravisensing and actin patterning defects that phenocopied the rmd mutant. Our findings outline a mechanism that links light signaling and gravity perception for straight shoot growth in rice.
Diatoms outcompete other phytoplankton for nitrate, yet little is known about the mechanisms underpinning this ability. Genomes and genome-enabled studies have shown that diatoms possess unique features of nitrogen metabolism however, the implications for nutrient utilization and growth are poorly understood. Using a combination of transcriptomics, proteomics, metabolomics, fluxomics, and flux balance analysis to examine short-term shifts in nitrogen utilization in the model pennate diatom in Phaeodactylum tricornutum, we obtained a systems-level understanding of assimilation and intracellular distribution of nitrogen. Chloroplasts and mitochondria are energetically integrated at the critical intersection of carbon and nitrogen metabolism in diatoms. Pathways involved in this integration are organelle-localized GS-GOGAT cycles, aspartate and alanine systems for amino moiety exchange, and a split-organelle arginine biosynthesis pathway that clarifies the role of the diatom urea cycle. This unique configuration allows diatoms to efficiently adjust to changing nitrogen status, conferring an ecological advantage over other phytoplankton taxa.
As limits on O2 availability during submergence impose severe constraints on aerobic respiration, the oxygen binding globin proteins of marine mammals are expected to have evolved under strong evolutionary pressures during their land-to-sea transition. Here, we address this question for the order Sirenia by retrieving, annotating, and performing detailed selection analyses on the globin repertoire of the extinct Steller’s sea cow (Hydrodamalis gigas), dugong (Dugong dugon), and Florida manatee (Trichechus manatus latirostris) in relation to their closest living terrestrial relatives (elephants and hyraxes). These analyses indicate most loci experienced elevated nucleotide substitution rates during their transition to a fully aquatic lifestyle. While most of these genes evolved under neutrality or strong purifying selection, the rate of nonsynonymous/synonymous replacements increased in two genes (Hbz-T1 and Hba-T1) that encode the α-type chains of hemoglobin (Hb) during each stage of life. Notably, the relaxed evolution of Hba-T1 is temporally coupled with the emergence of a chimeric pseudogene (Hba-T2/Hbq-ps) that contributed to the tandemly linked Hba-T1 of stem sirenians via interparalog gene conversion. Functional tests on recombinant Hb proteins from extant and ancestral sirenians further revealed that the molecular remodeling of Hba-T1 coincided with increased Hb–O2 affinity in early sirenians. Available evidence suggests that this trait evolved to maximize O2 extraction from finite lung stores and suppress tissue O2 offloading, thereby facilitating the low metabolic intensities of extant sirenians. In contrast, the derived reduction in Hb–O2 affinity in (sub)Arctic Steller’s sea cows is consistent with fueling increased thermogenesis by these once colossal marine herbivores.
Tea aroma is one of the most important factors affecting the character and quality of tea. Here we describe the practical application of methyl jasmonate (MeJA) to improve the aroma quality of teas. The changes of selected metabolites during crucial tea processing steps, namely, withering, fixing and rolling, and fermentation, were analyzed. MeJA treatment of tea leaves (12, 24, 48, and 168 h) greatly promotes the aroma quality of green, oolong, and black tea products when comparing with untreated ones (0 h) and as confirmed by sensory evaluation. MeJA modulates the aroma profiles before, during, and after processing. Benzyl alcohol, benzaldehyde, 2-phenylethyl alcohol, phenylacetaldehyde, and trans-2-hexenal increased 1.07- to 3-fold in MeJA-treated fresh leaves and the first two maintained at a higher level in black tea and the last two in green tea. This correlates with a decrease in aromatic amino acids by more than twofold indicating a direct relation to tryptophan- and phenylalanine-derived volatiles. MeJA-treated oolong tea was characterized by a more pleasant aroma. Especially the terpenoids linalool and oxides, geraniol, and carvenol increased by more than twofold.
Historically, the giant panda was widely distributed from northern China to southwestern Asia [1]. As a result of range contraction and fragmentation, extant individuals are currently restricted to fragmented mountain ranges on the eastern margin of the Qinghai-Tibet plateau, where they are distributed among three major population clusters [2]. However, little is known about the genetic consequences of this dramatic range contraction. For example, were regions where giant pandas previously existed occupied by ancestors of present-day populations, or were these regions occupied by genetically distinct populations that are now extinct? If so, is there any contribution of these extinct populations to the genomes of giant pandas living today? To investigate these questions, we sequenced the nuclear genome of an similar to 5,000-year-old giant panda from Jiangdongshan, Teng-chong County in Yunnan Province, China. We find that this individual represents a genetically distinct population that diverged prior to the diversification of modern giant panda populations. We find evidence of differential admixture with this ancient population among modern individuals originating from different populations as well as within the same population. We also find evidence for directional gene flow, which transferred alleles from the ancient population into the modern giant panda lineages. A variable proportion of the genomes of extant individuals is therefore likely derived from the ancient population represented by our sequenced individual. Although extant giant panda populations retain reasonable genetic diversity, our results suggest that this represents only part of the genetic diversity this species harbored prior to its recent range contractions.