Thiamine Synthesis Research Articles

Fusarium sacchari Effector FsMEP1 Contributes to Virulence by Disturbing Localization of Thiamine Thiazole Synthase ScTHI2 from Sugarcane

Fusarium sacchari is a significant pathogenic fungus that causes sugarcane Pokkah Boeng. Proteins secreted by pathogenic fungi can be delivered into hosts to suppress plant immunity and establish infection. However, there is still much to be discovered regarding F. sacchari’s secreted effectors in overcoming plant immunity. In this paper, we characterize a novel effector called FsMEP1, which is essential for the virulence of F. sacchari. FsMEP1 contains a conserved zinc-binding motif sequence, HEXXH, and is highly expressed during host infection. Using the Agrobacterium tumefaciens-mediated transient expression system, it was confirmed that FsMEP1 could suppress Bcl-2-associated X protein (BAX)-triggered cell death, callose deposition, and ROS explosion in Nicotiana benthamiana. Furthermore, the deletion of FsMEP1 demonstrated its requirement for contributing to the pathogenicity of F. sacchari in sugarcane. Further analysis revealed that FsMEP1 could interact with the sugarcane thiamine thiazole synthase ScTHI2 and disrupt its normal localization, thereby inhibiting the synthesis of thiamine and the defense responses mediated by ScTHI2. Based on these findings, we propose that ScTHI2 represents a potential molecular target for improving sugarcane resistance to Pokkah Boeng disease.

Thiamine deficiency underlies persistent gut dysbiosis and inflammation in people living with HIV on antiretroviral therapy

BackgroundAntiretroviral therapy (ART) has significantly improved the prognosis for people living with human immunodeficiency virus (PLWH). However, alterations in the gut microbiota of PLWH affect their metabolic environment and may contribute to chronic inflammation and premature aging, despite effective ART. Given that inflammation in the body have been shown to be risk factors for age-related diseases, elucidating changes in the gut microbiota and metabolic levels over time in individuals would be for understanding the dynamics in the body that lead to disease development.MethodsThe study assessed changes in blood markers, gut microbiota, and metabolic environment of PLWH on ART for four years, with participants divided into two groups based on median CD4 cell count (580/µL).ResultsThe high-CD4 group was more obese than the low-CD4 group, but there was no significant difference in comorbidities, such as diabetes, hypertension, and dyslipidemia. An investigation of the alterations in gut microbiota over a 4-year period revealed a reduction in specific groups of bacteria that produce short-chain fatty acids (SCFAs) and an increase in the occurrence of Enterobacteriaceae. In addition, a decline in bacterial thiamine synthesis was predicted, and a decreased abundance of the Ruminococcus gauvreauii group, which relies on supplementation with thiamine from symbiotic bacteria, was observed. The abundance of Collinsella and Catenibacterium at baseline was positively correlated with the predicted number of thiamine-synthesizing pathway. The presence of Catenibacterium at follow-up was inversely correlated with the rate of increase in plasma levels of lipopolysaccharide-binding protein, an indicator of a leaky gut. In terms of assessing the diversity of gut microbiota, a trend towards an increase in opportunistic bacteria with different genetic distances was observed in the high-CD4 group at follow-up.ConclusionThese observations suggested that, despite effective ART, the thiamine-deficient environment in the gut continues to reduce thiamine-requiring bacteria, such as certain SCFA-producing bacteria. As a result, secondary effects, such as proliferation of opportunistic bacteria and chronic inflammation associated with increased intestinal permeability, proceed in PLWH. Thus, certain essential nutrient deficiencies for gut metabolic environment may disrupt the symbiotic relationship between gut bacteria and increase the risk of developing inflammatory diseases.

Comparative Genomic and Genetic Evidence on a Role for the OarX Protein in Thiamin Salvage.

Salvage pathways for thiamin and its thiazole and pyrimidine moieties are poorly characterized compared to synthesis pathways. A candidate salvage gene is oarX, which encodes a short-chain dehydrogenase/reductase. In diverse bacteria, oarX clusters on the chromosome with genes of thiamin synthesis, salvage, or transport and is preceded by a thiamin pyrophosphate riboswitch. Thiamin and its moieties can undergo oxidations that convert a side-chain hydroxymethyl group to a carboxyl group, or the thiazole ring to a thiazolone, causing a loss of biological activity. To test if OarX participates in salvage of the carboxyl or thiazolone products, we used a genetic approach in Corynebacterium glutamicum ATCC 14067, which is auxotrophic for thiamin's pyrimidine moiety. This strain could not utilize the pyrimidine carboxyl derivative. This excluded a role in salvaging this product and narrowed the function search to metabolism of the carboxyl or thiazolone derivatives of thiamin or its thiazole moiety. However, a ΔthiG (thiazole auxotroph) strain was not rescued by any of these derivatives. Nor did deleting oarX affect rescue by the physiological pyrimidine and thiazole precursors of thiamin. These findings reinforce the genomic evidence that OarX has a function in thiamin metabolism and rule out five logical possibilities for what this function is.

Molecular mechanism of thiamine in mitigating drought stress in Chinese wingnut (Pterocarya stenoptera): Insights from transcriptomics

Urban garden plants are frequently affected by drought, which can hinder their growth, development, and greening effect. Previous studies have indicated that Chinese wingnut (Pterocarya stenoptera) responds to drought stress by increasing the expression of thiamine synthesis genes. In this study, it was found that exogenous thiamine can effectively alleviate the negative effects of drought stress on plants. Forward transcriptome sequencing and physiological tests were further conducted to reveal the molecular mechanism of thiamine in alleviating drought stress. Results showed that exogenous thiamine activated the expression of eight chlorophyll synthesis genes in Chinese wingnut under drought stress. Moreover, physiological indicators proved that chlorophyll content increased in leaves of Chinese wingnut with thiamine treatment under drought stress. Photosynthesis genes were also activated in Chinese wingnut treated with exogenous thiamine under drought stress, as supported by photosynthetic indicators PIabs and PItotal. Additionally, exogenous thiamine stimulated the expression of genes in the auxin-activated signaling pathway, thus attenuating the effects of drought stress. This study demonstrates the molecular mechanism of thiamine in mitigating the effects of drought stress on non-model woody plants lacking transgenic systems. This study also provides an effective method to mitigate the negative impacts of drought stress on plants.

Effects of long-term herbaceous plant restoration on microbial communities and metabolic profiles in coal gangue-contaminated soil

The soil microbial diversity in the gangue accumulation area is severely stressed by a variety of heavy metals, while the influence of long-term recovery of herbaceous plants on the ecological structure of gangue-contaminated soil is to be explored. Therefore, we analysed the differences in physicochemical properties, elemental changes, microbial community structure, metabolites and expression of related pathways in soils in the 10- and 20-year herbaceous remediation areas of coal gangue. Our results showed that phosphatase, soil urease, and sucrase activities of gangue soils significantly increased in the shallow layer after herbaceous remediation. However, in zone T1 (10-year remediation zone), the contents of harmful elements, such as Thorium (Th; 1.08-fold), Arsenic (As; 0.78-fold), lead (Pb; 0.99-fold), and uranium (U; 0.77-fold), increased significantly, whereas the soil microbial abundance and diversity also showed a significant decreasing trend. Conversely, in zone T2 (20-year restoration zone), the soil pH significantly increased by 1.03- to 1.06-fold and soil acidity significantly improved. Moreover, the abundance and diversity of soil microorganisms increased significantly, the expression of carbohydrates in soil was significantly downregulated, and sucrose content was significantly negatively correlated with the abundance of microorganisms, such as Streptomyces. A significant decrease in heavy metals was observed in the soil, such as U (1.01- to 1.09-fold) and Pb (1.13- to 1.25-fold). Additionally, the thiamin synthesis pathway was inhibited in the soil of the T1 zone; the expression level of sulfur (S)-containing histidine derivatives (Ergothioneine) was significantly up-regulated by 0.56-fold in the shallow soil of the T2 zone; and the S content in the soil significantly reduced. Aromatic compounds were significantly up-regulated in the soil after 20 years of herbaceous plant remediation in coal gangue soil, and microorganisms (Sphingomonas) with significant positive correlations with benzene ring-containing metabolites, such as Sulfaphenazole, were identified.

Functional and comparative analysis of THI1 gene in grasses with a focus on sugarcane.

De novo synthesis of thiamine (vitamin B1) in plants depends on the action of thiamine thiazole synthase, which synthesizes the thiazole ring, and is encoded by the THI1 gene. Here, we investigated the evolution and diversity of THI1 in Poaceae, where C4 and C3 photosynthetic plants co-evolved. An ancestral duplication of THI1 is observed in Panicoideae that remains in many modern monocots, including sugarcane. In addition to the two sugarcane copies (ScTHI1-1 and ScTHI1-2), we identified ScTHI1-2 alleles showing differences in their sequence, indicating divergence between ScTHI1-2a and ScTHI1-2b. Such variations are observed only in the Saccharum complex, corroborating the phylogeny. At least five THI1 genomic environments were found in Poaceae, two in sugarcane, M. sinensis, and S. bicolor. The THI1 promoter in Poaceae is highly conserved at 300 bp upstream of the start codon ATG and has cis-regulatory elements that putatively bind to transcription factors associated with development, growth, development and biological rhythms. An experiment set to compare gene expression levels in different tissues across the sugarcane R570 life cycle showed that ScTHI1-1 was expressed mainly in leaves regardless of age. Furthermore, ScTHI1 displayed relatively high expression levels in meristem and culm, which varied with the plant age. Finally, yeast complementation studies with THI4-defective strain demonstrate that only ScTHI1-1 and ScTHI1-2b isoforms can partially restore thiamine auxotrophy, albeit at a low frequency. Taken together, the present work supports the existence of multiple origins of THI1 harboring genomic regions in Poaceae with predicted functional redundancy. In addition, it questions the contribution of the levels of the thiazole ring in C4 photosynthetic plant tissues or potentially the relevance of the THI1 protein activity.

Dietary factors potentially impacting thiaminase I-mediated thiamine deficiency

Fish population declines from thiamine (vitamin B1) deficiency have been widespread in ecologically and economically valuable organisms, ranging from the Great Lakes to the Baltic Sea and, most recently, the California coast. Thiamine deficiencies in predatory fishes are often attributed to a diet of prey fishes with high levels of thiamine-degrading (e.g., thiaminase) enzymes, such as alewives, rainbow smelt, and anchovies. Since their discovery, thiaminase I enzymes have been recognized for breaking down thiamine into its pyrimidine and thiazole moieties using various nucleophilic co-substrates to afford cleavage, but these studies have not thoroughly considered other factors that could modify enzyme activity. We found the thiaminase I enzyme from Clostridium botulinum efficiently degrades thiamine in the presence of pyridoxine (vitamin B6) as a co-substrate but has relatively limited activity in the presence of nicotinic acid (vitamin B3). Using fluorescence measurements, thiamine degradation in an over-the-counter complete multivitamin formulation was inhibited, and a B-complex formulation required co-substrate supplementation for maximal thiamine depletion. These studies prompted the evaluation of specific constituents contributing to thiaminase I inhibition by both chromatography and fluorescence assays: Cu2+ potently and irreversibly inhibited thiamine degradation; ascorbic acid was a strong but reversible inhibitor; Fe2+, Mn2+ and Fe3+ modulated thiamine degradation to a lesser degree. The enhancement by pyridoxine and inhibition by Cu2+ extended to thiaminase-mediated degradation from Burkholderia thailandensis, Paenibacillus thiaminolyticus, and Paenibacillus apiarius in tryptic soy broth supernatants. These co-substrate limitations and the common presence of inhibitory dietary factors complement recent studies reporting that the intended function of thiaminase enzymes is to recycle thiamine breakdown products for thiamine synthesis, not thiamine degradation.

Directed Evolution of Aerotolerance in Sulfide-Dependent Thiazole Synthases.

Sulfide-dependent THI4 thiazole synthases could potentially be used to replace plant cysteine-dependent suicide THI4s, whose high protein turnover rates make thiamin synthesis exceptionally energy-expensive. However, sulfide-dependent THI4s are anaerobic or microoxic enzymes and hence unadapted to the aerobic conditions in plants; they are also slow enzymes (kcat < 1 h-1). To improve aerotolerance and activity, we applied continuous directed evolution under aerobic conditions in the yeast OrthoRep system to two sulfide-dependent bacterial THI4s. Seven beneficial single mutations were identified, of which five lie in the active-site cleft predicted by structural modeling and two recapitulate features of naturally aerotolerant THI4s. That single mutations gave substantial improvements suggests that further advance under selection will be possible by stacking mutations. This proof-of-concept study established that the performance of sulfide-dependent THI4s in aerobic conditions is evolvable and, more generally, that yeast OrthoRep provides a plant-like bridge to adapt nonplant enzymes to work better in plants.

A thiamine transporter is required for biofilm formation by Rhizobium sp. IRBG74.

Rhizobium sp. IRBG74 is a nitrogen fixing symbiont of Sesbania cannabina and a growth promoting endophyte of rice thus making it a good model to compare rhizobial interactions with legumes and cereals. In this report, we show that Rhizobium sp. IRBG74 forms biofilms on the roots of S. cannabina and rice. A mutant defective in biofilm formation was identified by screening a transposon mutant library. The transposon insertion was in thiQ, part of the thiBPQ operon that encodes the components of a thiamine/thiamine pyrophosphate ABC transporter. Complementation with thiBPQ partially restored biofilm formation. Addition of thiamine in growth media led to repression of thiC expression in the wild type strain but not in the thiQ mutant. These results suggest that thiBPQ is involved in thiamine/TPP transport in Rhizobium sp. IRBG74. Using a GUS reporter, we show that the expression of thiC is significantly higher in biofilm as compared to cells in planktonic growth. Based on these results we propose that Rhizobium sp. IRBG74 is thiamine limited and requires thiamine transport for efficient biofilm formation and plant colonization. Thiamine synthesis in aerobic bacteria such as Rhizobium requires O2 and thus could be inhibited in the microaerobic/anaerobic conditions in biofilms.

Population genomics of Australian indigenous Mesorhizobium reveals diverse nonsymbiotic genospecies capable of nitrogen-fixing symbioses following horizontal gene transfer.

Mesorhizobia are soil bacteria that establish nitrogen-fixing symbioses with various legumes. Novel symbiotic mesorhizobia frequently evolve following horizontal transfer of symbiosis-gene-carrying integrative and conjugative elements (ICESyms) to indigenous mesorhizobia in soils. Evolved symbionts exhibit a wide range in symbiotic effectiveness, with some fixing nitrogen poorly or not at all. Little is known about the genetic diversity and symbiotic potential of indigenous soil mesorhizobia prior to ICESym acquisition. Here we sequenced genomes of 144 Mesorhizobium spp. strains cultured directly from cultivated and uncultivated Australian soils. Of these, 126 lacked symbiosis genes. The only isolated symbiotic strains were either exotic strains used previously as legume inoculants, or indigenous mesorhizobia that had acquired exotic ICESyms. No native symbiotic strains were identified. Indigenous nonsymbiotic strains formed 22 genospecies with phylogenomic diversity overlapping the diversity of internationally isolated symbiotic Mesorhizobium spp. The genomes of indigenous mesorhizobia exhibited no evidence of prior involvement in nitrogen-fixing symbiosis, yet their core genomes were similar to symbiotic strains and they generally lacked genes for synthesis of biotin, nicotinate and thiamine. Genomes of nonsymbiotic mesorhizobia harboured similar mobile elements to those of symbiotic mesorhizobia, including ICESym-like elements carrying aforementioned vitamin-synthesis genes but lacking symbiosis genes. Diverse indigenous isolates receiving ICESyms through horizontal gene transfer formed effective symbioses with Lotus and Biserrula legumes, indicating most nonsymbiotic mesorhizobia have an innate capacity for nitrogen-fixing symbiosis following ICESym acquisition. Non-fixing ICESym-harbouring strains were isolated sporadically within species alongside effective symbionts, indicating chromosomal lineage does not predict symbiotic potential. Our observations suggest previously observed genomic diversity amongst symbiotic Mesorhizobium spp. represents a fraction of the extant diversity of nonsymbiotic strains. The overlapping phylogeny of symbiotic and nonsymbiotic clades suggests major clades of Mesorhizobium diverged prior to introduction of symbiosis genes and therefore chromosomal genes involved in symbiosis have evolved largely independent of nitrogen-fixing symbiosis.

The Vital Role of ShTHIC from the Endophyte OsiSh-2 in Thiamine Biosynthesis and Blast Resistance in the OsiSh-2-Rice Symbiont.

Endophytes can benefit the growth and stress resistance of host plants by secreting bioactive components. Thiamine is an essential vitamin involved in many metabolic pathways and can only be synthesized by microbes and plants. In this study, we found that thiamine could inhibit the development of the phytopathogen Magnaporthe oryzae and decrease the rice blast index under field conditions. In the thiamine biosynthesis pathway, the key enzyme ShTHIC of an endophyte Streptomyces hygroscopicus OsiSh-2 and OsTHIC of rice (Oryza sativa) were highly homologous. Gene overexpression or knockout approaches revealed that both THIC contributed to thiamine synthesis and resistance to M. oryzae. Furthermore, S. hygroscopicus OsiSh-2 colonization led to a decrease in the thiamine synthesis level of rice but still maintained thiamine homeostasis in rice. However, inoculation with the ShTHIC knockout strain ΔTHIC reduced the thiamine content in rice, although the thiamine synthesis level of rice was increased. After infection with M. oryzae, blast resistance was dramatically improved in OsiSh-2-inoculated rice but decreased in ΔTHIC-inoculated rice compared with non-inoculated rice. This result demonstrated that ShTHIC could regulate thiamine biosynthesis and consequently assist blast resistance in the OsiSh-2-rice symbiont. Our results revealed a novel blast-resistance mechanism mediated by a key thiamine biosynthetic enzyme from an endophyte OsiSh-2.

Thiamine functions as a key activator for modulating plant health and broad-spectrum tolerance in cotton.

Global climate changes cause an increase of abiotic and biotic stresses that tremendously threaten the world's crop security. However, studies on broad-spectrum response pathways involved in biotic and abiotic stresses are relatively rare. Here, by comparing the time-dependent transcriptional changes and co-expression analysis of cotton (Gossypium hirsutum) root tissues under abiotic and biotic stress conditions, we discovered the common stress-responsive genes and stress metabolism pathways under different stresses, which included the circadian rhythm, thiamine and galactose metabolism, carotenoid, phenylpropanoid, flavonoid, and zeatin biosynthesis, and the mitogen-activated protein kinase signaling pathway. We found that thiamine metabolism was an important intersection between abiotic and biotic stresses; the key thiamine synthesis genes, GhTHIC and GhTHI1, were highly induced at the early stage of stresses. We confirmed that thiamine was crucial and necessary for cotton growth and development, and its deficiency could be recovered by exogenous thiamine supplement. Furthermore, we revealed that exogenous thiamine enhanced stress tolerance in cotton via increasing calcium signal transduction and activating downstream stress-responsive genes. Overall, our studies demonstrated that thiamine played a crucial role in the tradeoff between plant health and stress resistance. The thiamine deficiency caused by stresses could transiently induce upregulation of thiamine biosynthetic genes in vivo, while it could be totally salvaged by exogenous thiamine application, which could significantly improve cotton broad-spectrum stress tolerance and enhance plant growth and development.

Discovery of Early-Branching Wolbachia Reveals Functional Enrichment on Horizontally Transferred Genes.

Wolbachia is a widespread endosymbiont of insects and filarial nematodes that profoundly influences host biology. Wolbachia has also been reported in rhizosphere hosts, where its diversity and function remain poorly characterized. The discovery that plant-parasitic nematodes (PPNs) host Wolbachia strains with unknown roles is of interest evolutionarily, ecologically, and for agriculture as a potential target for developing new biological controls. The goal of this study was to screen communities for PPN endosymbionts and analyze genes and genomic patterns that might indicate their role. Genome assemblies revealed 1 out of 16 sampled sites had nematode communities hosting a Wolbachia strain, designated wTex, that has highly diverged as one of the early supergroup L strains. Genome features, gene repertoires, and absence of known genes for cytoplasmic incompatibility, riboflavin, biotin, and other biosynthetic functions placed wTex between mutualist C + D strains and reproductive parasite A + B strains. Functional terms enriched in group L included protoporphyrinogen IX, thiamine, lysine, fatty acid, and cellular amino acid biosynthesis, while dN/dS analysis suggested the strongest purifying selection on arginine and lysine metabolism, and vitamin B6, heme, and zinc ion binding, suggesting these as candidate roles in PPN Wolbachia. Higher dN/dS pathways between group L, wPni from aphids, wFol from springtails, and wCfeT from cat fleas suggested distinct functional changes characterizing these early Wolbachia host transitions. PPN Wolbachia had several putative horizontally transferred genes, including a lysine biosynthesis operon like that of the mitochondrial symbiont Midichloria, a spirochete-like thiamine synthesis operon shared only with wCfeT, an ATP/ADP carrier important in Rickettsia, and a eukaryote-like gene that may mediate plant systemic acquired resistance through the lysine-to-pipecolic acid system. The Discovery of group L-like variants from global rhizosphere databases suggests diverse PPN Wolbachia strains remain to be discovered. These findings support the hypothesis of plant-specialization as key to shaping early Wolbachia evolution and present new functional hypotheses, demonstrating promise for future genomics-based rhizosphere screens.

Rapid Metabolism of 1,4-Dioxane to below Health Advisory Levels by Thiamine-Amended Rhodococcus ruber Strain 219

Bioremediation is a promising treatment technology for 1,4-dioxane-contaminated groundwater. However, metabolic dioxane-degrading bacteria identified to date are limited by their slow kinetics and inability to sustain growth at low dioxane concentrations (<100 μg/L). Furthermore, strains may underperform because of missing growth factors, such as amino acids or vitamins. In this work, we reevaluate Rhodococcus ruber strain 219 as a dioxane-degrading strain with bioaugmentation potential. We report rapid growth and metabolic dioxane degradation by R. ruber 219 when supplemented with thiamine (vitamin B1). We also discern that the strain lacks a complete de novo thiamine synthesis pathway, indicating that R. ruber 219 is a probable thiamine auxotroph. However, when supplemented with thiamine, the strain’s Monod kinetics (Ks = 0.015 ± 0.03 μg/L) and exceedingly low Smin (0.49 ± 1.16 μg/L) suggest this strain can maintain growth at very low dioxane concentrations (<100 μg/L). Accordingly, we demonstrate that thiamine-grown R. ruber 219 sustains degradation of dilute dioxane (<100 μg/L) to below health advisory levels. This is the first study to report sustained metabolic dioxane biodegradation to below health advisory levels of 0.35 μg/L. Overall, our findings solidify R. ruber 219 as a promising candidate for bioremediation of dioxane-contaminated groundwater.

Ontogenetic Dietary Changes and Thiamine Status of Lake Ontario Chinook Salmon, 2005–2006

AbstractIn the Great Lakes, an Alewife Alosa pseudoharengus diet has been reported to cause thiamine deficiency in salmonines as a result of high thiaminase activity. The ontogeny of thiamine deficiency and its relationship to Alewife consumption have not been determined in Chinook Salmon Oncorhynchus tshawytscha within this system. Using stable isotope mixing models along with muscle thiamine, we assessed ontogenetic changes in the diet and corresponding muscle thiamine status of Lake Ontario Chinook Salmon ranging in TL from 100 to 1,000 mm. Thiamine levels were highest in young of the year collected in a tributary of Lake Ontario but declined once Chinook Salmon entered Lake Ontario. In Lake Ontario, the diets of juvenile (200–400 mm; 97–99%) and adult (&gt;400 mm; 97–100%) Chinook Salmon consisted almost entirely of Alewives as estimated using mixing models. This Alewife diet was associated with an ontogenetic decline in muscle thiamine among Chinook Salmon. The asymptotic decline in thiamine concentration with increasing size plateaued at 720–758 pmol/g of muscle. The proportion of individuals below a thiamine threshold of 500 pmol/g (associated with a loss of equilibrium) increased with TL up to 799 mm and then declined between 800 and 1,000 mm. At these muscle concentrations, no outward signs of thiamine deficiency were observed. Calculation of total mass accumulation of body stores of thiamine (muscle concentration times body weight) showed an unexpected and significant increase with size. Although it appears that dietary Alewives affected thiamine status, there were undetermined factors, such as incomplete destruction of thiamine by thiaminase, gut synthesis of thiamine, gill absorption of water thiamine, and/or other factors, that contributed to the overall thiamine status of these Chinook Salmon.

Melatonin elevated Sclerotinia sclerotiorum resistance via modulation of ATP and glucosinolate biosynthesis in Brassica rapa ssp. pekinensis

Sclerotinia stem rot is a common disease found in Brassica rapa that is caused by the necrotic plant pathogen Sclerotinia sclerotiorum. Melatonin (MT) has known biological activity and effectively relieved this type of Sclerotinia stem rot in B. rapa. To better understand the mechanisms behind MT-induced S. sclerotiorum resistance in B. rapa, we performed both proteomic and metabolomic analysis. Our results showed that during S. sclerotiorum infection, thiamine synthesis was activated and defended against it. In infected leaves, ribosomal synthesis-related proteins responded positively to MT treatment. Integrated proteomic and metabolomic analysis showed that amino acid metabolism was activated by MT treatment. After MT treatment, adenosine-triphosphate (ATP) content and the activity of antioxidant enzymes were both increased in B. rapa infected leaves. Cysteine synthase, sulfur transfer-related proteins, and glucosinolate (GS) were all increased after MT treatment in infected B. rapa leaves. Taken together, these results indicated that B. rapa leaves promoted thiamine formation to defend against S. sclerotiorum infection. Moreover, MT helped further induce antioxidant activation in B. rapa in an ATP-dependent manner and stimulating GS biosynthesis to well inhibit the S. sclerotiorum infection. SignificanceMelatonin (MT) has biological activity and effectively relieved the Sclerotinia stem rot of Brassica rapa caused by the necrotic plant pathogen Sclerotinia sclerotiorum. In order to reveal the molecular mechanisms of MT-induced S. sclerotiorum resistance in B. rapa, comprehensive proteomic and metabolomic analyses were conducted. The integration analysis of omic-data illustrated that the modulation of ATP and glucosinolate biosynthesis induced by MT administration helped to defend the infection of S. sclerotiorum in B. rapa. Our results will provide insights into MT-induced anti-fungal mechanism and therapeutic strategies to mitigate Sclerotinia stem rot of B. rapa, thereby increasing plant yield and decreasing economic losses.

Ensemble Switching Unveils a Kinetic Rheostat Mechanism of the Eukaryotic Thiamine Pyrophosphate Riboswitch.

Thiamine pyrophosphate (TPP) riboswitches regulate thiamine metabolism by inhibiting the translation of enzymes essential to thiamine synthesis pathways upon binding to thiamine pyrophosphate in cells across all domains of life. Recent work on the Arabidopsis thaliana TPP riboswitch suggests a multistep TPP binding process involving multiple riboswitch configurational ensembles and Mg2+ dependence underlies the mechanism of TPP recognition and subsequent transition to the expression-inhibiting state of the aptamer domain followed by changes in the expression platform. However, details of the relationship between TPP riboswitch conformational changes and interactions with TPP and Mg2+ in the aptamer domain constituting this mechanism are unknown. Therefore, we integrated single-molecule multiparameter fluorescence and force spectroscopy with atomistic molecular dynamics simulations and found that conformational transitions within the aptamer domain's sensor helices associated with TPP and Mg2+ ligand binding occurred between at least five different ensembles on timescales ranging from µs to ms. These dynamics are orders of magnitude faster than the 10 sec-timescale folding kinetics associated with expression-state switching in the switch helix. Together, our results show that a TPP and Mg2+ dependent mechanism determines dynamic configurational state ensemble switching of the aptamer domain's sensor helices that regulate the switch helix's stability, which ultimately may lead to the expression-inhibiting state of the riboswitch. Additionally, we propose that two pathways exist for ligand recognition and that this mechanism underlies a kinetic rheostat-like behavior of the Arabidopsis thaliana TPP riboswitch.

Occurrence, Evolution and Specificities of Iron-Sulfur Proteins and Maturation Factors in Chloroplasts from Algae.

Iron-containing proteins, including iron-sulfur (Fe-S) proteins, are essential for numerous electron transfer and metabolic reactions. They are present in most subcellular compartments. In plastids, in addition to sustaining the linear and cyclic photosynthetic electron transfer chains, Fe-S proteins participate in carbon, nitrogen, and sulfur assimilation, tetrapyrrole and isoprenoid metabolism, and lipoic acid and thiamine synthesis. The synthesis of Fe-S clusters, their trafficking, and their insertion into chloroplastic proteins necessitate the so-called sulfur mobilization (SUF) protein machinery. In the first part, we describe the molecular mechanisms that allow Fe-S cluster synthesis and insertion into acceptor proteins by the SUF machinery and analyze the occurrence of the SUF components in microalgae, focusing in particular on the green alga Chlamydomonas reinhardtii. In the second part, we describe chloroplastic Fe-S protein-dependent pathways that are specific to Chlamydomonas or for which Chlamydomonas presents specificities compared to terrestrial plants, putting notable emphasis on the contribution of Fe-S proteins to chlorophyll synthesis in the dark and to the fermentative metabolism. The occurrence and evolutionary conservation of these enzymes and pathways have been analyzed in all supergroups of microalgae performing oxygenic photosynthesis.

Essential Amino Acid Enrichment and Positive Selection Highlight Endosymbiont's Role in a Global Virus-Vectoring Pest.

Host-associated microbes display remarkable convergence in genome repertoire resulting from selection to supplement missing host functions. Nutritional supplementation has been proposed in the verrucomicrobial endosymbiont Xiphinematobacter sp., which lives within a globally widespread group of plant-parasitic nematodes that vector damaging nepoviruses to plants. Only one genome sequence has been published from this symbiont, leaving unanswered questions about its diversity, host range, role, and selective pressures within its hosts. Because its hosts are exceptionally resistant to culturing, this symbiont is best studied through advanced genomic approaches. To analyze the role of Xiphinematobacter sp. in its host, sequencing was performed on nematode communities, and then genomes were extracted for comparative genomics, gene ontology enrichment tests, polymorphism analysis, de Bruijn-based genome-wide association studies, and tests of pathway- and site-specific selection on genes predicted play a role in the symbiosis. Results showed a closely clustered set of Xiphinematobacter isolates with reduced genomes of ∼917 kbp, for which a new species was proposed. Symbionts shared only 2.3% of genes with outgroup Verrucomicrobia, but comparative analyses showed high conservation of all 10 essential amino acid (EAA) biosynthesis pathways plus several vitamin pathways. These findings were supported by gene ontology enrichment tests and high polymorphisms in these pathways compared with background. Genome-wide association analysis confirmed high between-species fixation of alleles with significant functional enrichment for EAA and thiamine synthesis. Strong positive selection was detected on sites within these pathways, despite several being under increased purifying selection. Together, these results suggest that supplementation of EAAs missing in the host diet may drive this widespread symbiosis.IMPORTANCE Xiphinematobacter spp. are distinctly evolved intracellular symbionts in the phylum Verrucomicrobia, which includes the important human gut-associated microbe Akkermansia muciniphila and many highly abundant free-living soil microbes. Like Akkermansia sp., Xiphinematobacter sp. is obligately associated with the gut of its hosts, which in this case consists of a group of plant-parasitic nematodes that are among the top 10 most destructive species to global agriculture, by vectoring plant viruses. This study examined the hypothesis that the key to this symbiont's stable evolutionary association with its host is through provisioning nutrients that its host cannot make that may be lacking in the nematode's plant phloem diet, such as essential amino acids and several vitamins. The significance of our research is in demonstrating, using population genomics, the signatures of selective pressure on these hypothesized roles to ultimately learn how this independently evolved symbiont functionally mirrors symbionts of phloem-feeding insects.

Thiamine: a key nutrient for yeasts during wine alcoholic fermentation.

Alcoholic fermentation is a crucial step of winemaking, during which yeasts convert sugars to alcohol and also produce or biotransform numerous flavour compounds. In this context, nutrients are essential compounds to support yeast growth and ultimately ensure complete fermentation, as well as optimized production of flavour compounds over that of off-flavour compounds. In particular, the vitamin thiamine not only plays an essential cofactor role for several enzymes involved in various metabolic pathways, including those leading to the production of wine-relevant flavour compounds, but also aids yeast survival via thiamine-dependent stress protection functions. Most yeast species are able to both assimilate exogenous thiamine into the cell and synthesize thiamine de novo. However, the mechanism and level of thiamine accumulation depend on several factors. This review provides an in-depth overview of thiamine utilization and metabolism in the model yeast species Saccharomyces cerevisiae, as well as the current knowledge on (1) the intracellular functions of thiamine, (2) the balance between and regulation of uptake and synthesis of thiamine and (3) the multitude of factors influencing thiamine availability and utilization. For the latter, a particular emphasis is placed on conditions occurring during wine fermentation. The adequacy of thiamine concentration in grape must to ensure successful fermentation is discussed together with the effect of thiamine concentration on fermentation kinetics and on wine sensory properties. This knowledge may serve as a resource to optimise thiamine concentrations for optimal industrial application of yeasts. KEY POINTS: • Thiamine uptake is preferred over biosynthesis and is transcriptionally repressed. • Multiple factors affect thiamine synthesis, availability and uptake for wine yeast. • Thiamine availability impacts fermentation kinetics and wine's sensory properties.