C4 Metabolism Research Articles

Photosynthetic Electron Flows and Networks of Metabolite Trafficking to Sustain Metabolism in Photosynthetic Systems

Photosynthetic eukaryotes have metabolic pathways that occur in distinct subcellular compartments. However, because metabolites synthesized in one compartment, including fixed carbon compounds and reductant generated by photosynthetic electron flows, may be integral to processes in other compartments, the cells must efficiently move metabolites among the different compartments. This review examines the various photosynthetic electron flows used to generate ATP and fixed carbon and the trafficking of metabolites in the green alga Chlamydomomas reinhardtii; information on other algae and plants is provided to add depth and nuance to the discussion. We emphasized the trafficking of metabolites across the envelope membranes of the two energy powerhouse organelles of the cell, the chloroplast and mitochondrion, the nature and roles of the major mobile metabolites that move among these compartments, and the specific or presumed transporters involved in that trafficking. These transporters include sugar-phosphate (sugar-P)/inorganic phosphate (Pi) transporters and dicarboxylate transporters, although, in many cases, we know little about the substrate specificities of these transporters, how their activities are regulated/coordinated, compensatory responses among transporters when specific transporters are compromised, associations between transporters and other cellular proteins, and the possibilities for forming specific ‘megacomplexes’ involving interactions between enzymes of central metabolism with specific transport proteins. Finally, we discuss metabolite trafficking associated with specific biological processes that occur under various environmental conditions to help to maintain the cell’s fitness. These processes include C4 metabolism in plants and the carbon concentrating mechanism, photorespiration, and fermentation metabolism in algae.

Metabolic engineering of the serine/glycine network as a means to improve the nitrogen content of crops.

In plants, L-serine (Ser) biosynthesis occurs through various pathways and is highly dependent on the atmospheric CO2 concentration, especially in C3 species, due to the association of the Glycolate Pathway of Ser Biosynthesis (GPSB) with photorespiration. Characterization of a second plant Ser pathway, the Phosphorylated Pathway of Ser Biosynthesis (PPSB), revealed that it is at the crossroads of carbon, nitrogen, and sulphur metabolism. The PPSB comprises three sequential reactions catalysed by 3-phosphoglycerate dehydrogenase (PGDH), 3-phosphoSer aminotransferase (PSAT) and 3-phosphoSer phosphatase (PSP). PPSB was overexpressed in plants exhibiting two different modes of photosynthesis: Arabidopsis (C3 metabolism), and maize (C4 metabolism), under ambient (aCO2) and elevated (eCO2) CO2 growth conditions. Overexpression in Arabidopsis of the PGDH1 gene alone or PGDH1, PSAT1 and PSP1 in combination increased the Ser levels but also the essential amino acids threonine (aCO2), isoleucine, leucine, lysine, phenylalanine, threonine and methionine (eCO2) compared to the wild-type. These increases translated into higher protein levels. Likewise, starch levels were also increased in the PPSB-overexpressing lines. In maize, PPSB-deficient lines were obtained by targeting PSP1 using Cas9 endonuclease. We concluded that the expression of PPSB in maize male gametophyte is required for viable pollen development. Maize lines overexpressing the AtPGDH1 gene only displayed higher protein levels but not starch at both aCO2 and eCO2 conditions, this translated into a significant rise in the nitrogen/carbon ratio. These results suggest that metabolic engineering of PPSB in crops could enhance nitrogen content, particularly under upcoming eCO2 conditions where the activity of GPSB is limited.

Formaldehyde: An Essential Intermediate for C1 Metabolism and Bioconversion.

Formaldehyde is an intermediate metabolite of methylotrophic microorganisms that can be obtained from formate and methanol through oxidation-reduction reactions. Formaldehyde is also a one-carbon (C1) compound with high uniquely reactive activity and versatility, which is more amenable to further biocatalysis. Biosynthesis of high-value-added chemicals using formaldehyde as an intermediate is theoretically feasible and promising. This review focuses on the design of the biosynthesis of high-value-added chemicals using formaldehyde as an essential intermediate. The upstream biosynthesis and downstream bioconversion pathways of formaldehyde as an intermediate metabolite are described in detail, aiming to highlight the important role of formaldehyde in the transition from inorganic to organic carbon and carbon chain elongation. In addition, challenges and future directions of formaldehyde as an intermediate for the chemicals are discussed, with the expectation of providing ideas for the utilization of C1.

Identifying the botanical origin of alcohol using 2H SNIF NMR: A case study of “polish vodka” PGI

In this study, we utilized 2H SNIF NMR and chemometric techniques to differentiate the botanical origin of raw materials used in vodka production in Poland, specifically focusing on plants with C3 metabolism such as grain, potato, and sugar beet. Additionally, for the first time, mixtures of alcohols from different C3 plants were analysed to detect adulteration. Our goal was to determine if it is possible to detect the addition of a different raw material in vodka made from a mixture of alcohols derived from C3 plants. Significant isotopic differences were confirmed using analysis of variance and Tukey's tests. Linear relationships in grain-potato, grain-sugar beet, and beet-potato mixtures enabled composition determination. The detectability threshold for adulterants ranged from 10 % to 50 %, depending on the type of raw material. Our findings suggest that 2H SNIF-NMR is an effective tool for authenticating vodka and detecting adulteration in products marketed as “Polish vodka.”

Formate-tetrahydrofolate ligase: supplying the cytosolic one-carbon network in roots with one-carbon units originating from glycolate.

The metabolism of tetrahydrofolate (H4PteGlun)-bound one-carbon (C1) units (C1 metabolism) is multifaceted and required for plant growth, but it is unclear what of many possible synthesis pathways provide C1 units in specific organelles and tissues. One possible source of C1 units is via formate-tetrahydrofolate ligase, which catalyzes the reversible ATP-driven production of 10-formyltetrahydrofolate (10-formyl-H4PteGlun) from formate and tetrahydrofolate (H4PteGlun). Here, we report biochemical and functional characterization of the enzyme from Arabidopsis thaliana (AtFTHFL). We show that the recombinant AtFTHFL has lower Km and kcat values with pentaglutamyl tetrahydrofolate (H4PteGlu5) as compared to monoglutamyl tetrahydrofolate (H4PteGlu1), resulting in virtually identical catalytic efficiencies for the two substrates. Stable transformation of Arabidopsis plants with the EGFP-tagged AtFTHFL, followed with fluorescence microscopy, demonstrated cytosolic signal. Two independent T-DNA insertion lines with impaired AtFTHFL function had shorter roots compared to the wild type plants, demonstrating the importance of this enzyme for root growth. Overexpressing AtFTHFL led to the accumulation of H4PteGlun + 5,10-methylene-H4PteGlun and serine, accompanied with the depletion of formate and glycolate, in roots of the transgenic Arabidopsis plants. This metabolic adjustment supports the hypothesis that AtFTHFL feeds the cytosolic C1 network in roots with C1 units originating from glycolate, and that these units are then used mainly for biosynthesis of serine, and not as much for the biosynthesis of 5-methyl-H4PteGlun, methionine, and S-adenosylmethionine. This finding has implications for any future attempts to engineer one-carbon unit-requiring products through manipulation of the one-carbon metabolic network in non-photosynthetic organs.

Assessment of physio-morphological traits, genetic variability, and growth performance among amaranth (Amaranthus species) genotypes from Ethiopia

Amaranths are a type of plant that belongs to the NAD-malic enzyme-type C4 metabolism category. They have a unique C4 anatomy, which is present in their bracts, cotyledons, and leaves. This allows them to produce food through the C4 photosynthetic pathway and rapidly adapt to unfavorable environmental conditions. In this study, 120 amaranth genotypes were evaluated for physio-morphological traits, genetic variability, and growth performance assessment from Ethiopia. The results of the analysis of variance showed that all examined physio-morphological parameters, except the rate of photosynthesis and stomata conductance, had mean squares that varied considerably (P < 0.001) owing to genotypes. The estimates of genetic variability, heritability, and expected genetic advance indicated an incredible extent of genetic diversity among amaranth genotypes, with a significant selection pressure for these traits in the population to produce better genotypes for improved amaranth. Selection based on desirable features such as leaf-to-air vapor pressure deficit, transpiration rate, chlorophyll a, chlorophyll b, total chlorophyll, carotenoid, leaf area, plant height, leaf number, and root weight can be useful in achieving the intended genetic gains for improvement since these traits appear to be more controlled by additive gene activity. Thus, selection in amaranth genotypes may consider these desired yield-related features. Moreover, the study showed that certain genotypes (ALE-073) exhibited better intercellular CO2 concentration (Ci), leaf-to-air vapor pressure deficit (VPD), transpiration rate (E), and leaf number (LN), resulting in better grain yield. Understanding the relationship between LA and E can help in selecting crops for high E and may provide an avenue to improve leaf yield. Furthermore, some of the selected genotypes in this study could be used as potential parents for improving the genetic gain in amaranth breeding programs. The study concluded that there was additive gene action present since the Ch a, Ch b, TCh, and Tca markers exhibited 100 % heritability. This showed that the use of these characteristics for selection, which indicated a potentially exploitable variation, would be more effective and successful in the long run in breeding programs than the use of other traits for splitting generations.

Binding Sites of Bicarbonate in Phosphoenolpyruvate Carboxylase.

Phosphoenolpyruvate carboxylase (PEPC) is used in plant metabolism for fruit maturation or seed development as well as in the C4 and crassulacean acid metabolism (CAM) mechanisms in photosynthesis, where it is used for the capture of hydrated CO2 (bicarbonate). To find the yet unknown binding site of bicarbonate in this enzyme, we have first identified putative binding sites with nonequilibrium molecular dynamics simulations and then ranked these sites with alchemical free energy calculations with corrections of computational artifacts. Fourteen pockets where bicarbonate could bind were identified, with three having realistic binding free energies with differences with the experimental value below 1 kcal/mol. One of these pockets is found far from the active site at 14 Å and predicted to be an allosteric binding site. In the two other binding sites, bicarbonate is in direct interaction with the magnesium ion; neither sequence alignment nor the study of mutant K606N allowed to discriminate between these two pockets, and both are good candidates as the binding site of bicarbonate in phosphoenolpyruvate carboxylase.

Abstract 4730: Pharmacodynamic determinants of anti tumor activity of SHMT2 inhibitors

Abstract These studies investigate factors that impact the antitumor activity of SHMT2 targeting including drug accumulation, metabolism, expression of one carbon (C1) related enzymes, and tumor hypoxia. Mitochondrial C1 metabolism is upregulated in a variety of cancer types including leukemias and solid tumors. The mitochondrial C1 enzymes serine hydroxymethyltransferase (SHMT) 2 and 5,10-methylene tetrahydrofolate (THF) dehydrogenase 2 (MTHFD2) are among the top 5 overexpressed metabolic enzymes in tumors vs normal tissues in a wide range of tumor types. Thus, SHMT2 and MTHFD2 are potential therapeutic targets for cancer, sparing normal tissues that are less reliant on mitochondrial C1 metabolism. SHMT2 metabolizes serine and THF to produce glycine and 5,10-methylene THF; MTHFD2 metabolizes 5,10-methylene THF to NADH and 10-formyl THF which is converted to formate by MTHFD1L. Thus, mitochondrial C1 metabolism is the principal source of C1 units for cellular biosynthesis and provides the majority of glycine for protein, purine and glutathione synthesis. We previously discovered novel 5-substituted pyrrolo[3,2-d]pyrimidine antifolate compounds which exhibit primary inhibition at mitochondrial SHMT2 along with cytosolic SHMT1 and de novo purine biosynthesis at glycinamide ribonucleotide and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferases. The lead compound AGF347 is taken up into cells by the facilitative transporters reduced folate carrier (SLC19A1) and the proton coupled folate transporter (SLC46A1) and like folate cofactors is metabolized to polyglutamate (PG) forms by folylpolyglutamate synthase (FPGS) which promotes retention in cytosolic and mitochondrial compartments and target enzyme engagement. We investigated the impact of folate transporter expression on metabolism to PGs for SHMT2 targeted antifolates. Folate transporter expression inversely correlated with anti-tumor efficacy of SHMT2 targeted antifolates and overexpression of FPGS resulted in dramatically increased sensitivities. However, for pyrazolopyran inhibitors of SHMT2 (e.g. SHIN1) which enter cells by diffusion and are not metabolized to polyglutamates, increased transporter and FPGS levels dramatically decreased anti-tumor activity. As SHMT2 is a target of HIF1α, we studied the impact of multitargeting SHMT2 and purine biosynthesis by pyrrolo[3,2-d]pyrimidine antifolates under hypoxic conditions. Our results establish that multi-targeting SHMT2 and purine biosynthesis preserves anti-tumor activity under hypoxia. Collectively, our results demonstrate that SHMT2 targeted pyrrolo[3,2-d]pyrimidine antifolates exhibit increased antitumor efficacies in cells expressing low folate transporter expression and is further enhanced by increased FPGS activity. Additionally, we find multitargeting of SHMT2 and de novo purine biosynthesis manifests as antitumor activity even under hypoxic conditions. Citation Format: Mathew Joseph Schneider, Carrie O'Connor, Xun Bao, Md. Junayed Nayeen, Zhanjun Hou, Jing Li, Aleem Gangjee, Larry Matherly. Pharmacodynamic determinants of anti tumor activity of SHMT2 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4730.

Multiple Roles of Glycerate Kinase-From Photorespiration to Gluconeogenesis, C4 Metabolism, and Plant Immunity.

Plant glycerate kinase (GK) was previously considered an exclusively chloroplastic enzyme of the glycolate pathway (photorespiration), and its sole predicted role was to return most of the glycolate-derived carbon (as glycerate) to the Calvin cycle. However, recent discovery of cytosolic GK revealed metabolic links for glycerate to other processes. Although GK was initially proposed as being solely regulated by substrate availability, subsequent discoveries of its redox regulation and the light involvement in the production of chloroplastic and cytosolic GK isoforms have indicated a more refined regulation of the pathways of glycerate conversion. Here, we re-evaluate the importance of GK and emphasize its multifaceted role in plants. Thus, GK can be a major player in several branches of primary metabolism, including the glycolate pathway, gluconeogenesis, glycolysis, and C4 metabolism. In addition, recently, the chloroplastic (but not cytosolic) GK isoform was implicated as part of a light-dependent plant immune response to pathogen attack. The origins of glycerate are also discussed here; it is produced in several cell compartments and undergoes huge fluctuations depending on light/dark conditions. The recent discovery of the vacuolar glycerate transporter adds yet another layer to our understanding of glycerate transport/metabolism and that of other two- and three-carbon metabolites.

Mitochondrial and Cytosolic One-Carbon Metabolism Is a Targetable Metabolic Vulnerability in Cisplatin-Resistant Ovarian Cancer.

One-carbon (C1) metabolism is compartmentalized between the cytosol and mitochondria with the mitochondrial C1 pathway as the major source of glycine and C1 units for cellular biosynthesis. Expression of mitochondrial C1 genes including SLC25A32, serine hydroxymethyl transferase (SHMT) 2, 5,10-methylene tetrahydrofolate dehydrogenase 2, and 5,10-methylene tetrahydrofolate dehydrogenase 1-like was significantly elevated in primary epithelial ovarian cancer (EOC) specimens compared with normal ovaries. 5-Substituted pyrrolo[3,2-d]pyrimidine antifolates (AGF347, AGF359, AGF362) inhibited proliferation of cisplatin-sensitive (A2780, CaOV3, IGROV1) and cisplatin-resistant (A2780-E80, SKOV3) EOC cells. In SKOV3 and A2780-E80 cells, colony formation was inhibited. AGF347 induced apoptosis in SKOV3 cells. In IGROV1 cells, AGF347 was transported by folate receptor (FR) α. AGF347 was also transported into IGROV1 and SKOV3 cells by the proton-coupled folate transporter (SLC46A1) and the reduced folate carrier (SLC19A1). AGF347 accumulated to high levels in the cytosol and mitochondria of SKOV3 cells. By targeted metabolomics with [2,3,3-2H]L-serine, AGF347, AGF359, and AGF362 inhibited SHMT2 in the mitochondria. In the cytosol, SHMT1 and de novo purine biosynthesis (i.e., glycinamide ribonucleotide formyltransferase, 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase) were targeted; AGF359 also inhibited thymidylate synthase. Antifolate treatments of SKOV3 cells depleted cellular glycine, mitochondrial NADH and glutathione, and showed synergistic in vitro inhibition toward SKOV3 and A2780-E80 cells when combined with cisplatin. In vivo studies with subcutaneous SKOV3 EOC xenografts in SCID mice confirmed significant antitumor efficacy of AGF347. Collectively, our studies demonstrate a unique metabolic vulnerability in EOC involving mitochondrial and cytosolic C1 metabolism, which offers a promising new platform for therapy.

CLPP-Null Eukaryotes with Excess Heme Biosynthesis Show Reduced L-arginine Levels, Probably via CLPX-Mediated OAT Activation.

The serine peptidase CLPP is conserved among bacteria, chloroplasts, and mitochondria. In humans and mice, its loss causes Perrault syndrome, which presents with growth deficits, infertility, deafness, and ataxia. In the filamentous fungus Podospora anserina, CLPP loss leads to longevity. CLPP substrates are selected by CLPX, an AAA+ unfoldase. CLPX is known to target delta-aminolevulinic acid synthase (ALAS) to promote pyridoxal phosphate (PLP) binding. CLPX may also influence cofactor association with other enzymes. Here, the evaluation of P. anserina metabolomics highlighted a reduction in arginine/histidine levels. In Mus musculus cerebellum, reductions in arginine/histidine and citrulline occurred with a concomitant accumulation of the heme precursor protoporphyrin IX. This suggests that the increased biosynthesis of 5-carbon (C5) chain deltaALA consumes not only C4 succinyl-CoA and C1 glycine but also specific C5 delta amino acids. As enzymes responsible for these effects, the elevated abundance of CLPX and ALAS is paralleled by increased OAT (PLP-dependent, ornithine delta-aminotransferase) levels. Possibly as a consequence of altered C1 metabolism, the proteome profiles of P. anserina CLPP-null cells showed strong accumulation of a methyltransferase and two mitoribosomal large subunit factors. The reduced histidine levels may explain the previously observed metal interaction problems. As the main nitrogen-storing metabolite, a deficiency in arginine would affect the urea cycle and polyamine synthesis. Supplementation of arginine and histidine might rescue the growth deficits of CLPP-mutant patients.

Pushing the limits of C3 intrinsic water use efficiency in Mediterranean semiarid steppes: Responses of a drought‐avoider perennial grass to climate aridification

Abstract Intrinsic water use efficiency (WUEi) reflects the trade‐off between photosynthetic carbon gain and water loss through stomatal conductance and is key for understanding dryland plant responses to climate change. Stipa tenacissima is a perennial tussock C3 grass with an opportunistic, drought‐avoiding water use strategy that dominates arid and semiarid steppes across the western Mediterranean region. However, its ecophysiological responses to aridification and woody shrub encroachment, a major land‐use change in drylands worldwide, are not well‐understood. We investigated the variations in leaf stable isotopes (δ18O, δ13C, δ15N), nutrient concentrations (N, P, K), and culm water content and isotopic composition (δ18O, δ2H) of paired pure‐grass and shrub‐encroached S. tenacissima steppes along a 350 km aridity gradient in Spain (10 sites, 160 individuals). Culm water isotopes revealed that S. tenacissima is a shallow‐rooted grass that depends heavily on recent rainwater for water uptake, which may render it vulnerable to increasingly irregular rainfall combined with faster topsoil drying under climate warming and aridification. With increasing aridity, S. tenacissima enhanced leaf‐level WUEi through more stringent stomatal regulation of plant water flux and carbon assimilation (higher δ13C and δ18O), reaching exceptionally high δ13C values (−23‰ to −21‰) at the most arid steppes. Foliar N concentration was remarkably low across sites regardless of woody shrub encroachment, evidencing severe water and N co‐limitation of photosynthesis and productivity. Shrub encroachment decreased leaf P and K but did not affect S. tenacissima water status. Perennial grass cover decreased markedly with both declining winter rainfall and shrub encroachment suggesting population‐level rather than individual‐level responses of S. tenacissima to these changes. The fundamental physiological constraints of photosynthetic C3 metabolism combined with low foliar N content may hamper the ability of S. tenacissima and other drought‐avoider species with shallow roots to achieve further adaptive improvements in WUEi under increasing climatic stress. A drought‐avoiding water use strategy based on early stomatal closure and photosynthesis suppression during prolonged rainless periods may thus compromise the capacity of semiarid S. tenacissima steppes to maintain perennial grass cover, sustain productivity and cope with ongoing climate aridification at the drier parts of their current distribution. Read the free Plain Language Summary for this article on the Journal blog.

Regulatory effect of pipecolic acid (Pip) on the antioxidant system activity of Mesembryanthemum crystallinum plants exposed to bacterial treatment.

The presented study aims to elucidate the regulatory role of Pipecolic acid (Pip) in modulating the antioxidant system activity of Mesembryanthemum crystallinum plants exposed to Pseudomonas syringae infestation. M. crystallinum, known for its semi-halophytic nature, can transition its metabolism from C3 to CAM under salt stress conditions. The research encompasses the antioxidant system of the plants, covering both enzymatic and low molecular weight components. The findings indicate that Pip supplementation confers a beneficial effect on certain elements of the antioxidant system when the plants are subjected to stress induced by bacteria. Notably, during critical periods, particularly in the initial days post-bacterial treatment, M. crystallinum plants supplemented with Pip and exhibiting C3 metabolism display heightened total antioxidant capacity. This enhancement includes increased superoxide dismutase activity and elevated levels of glutathione and proline. However, in plants with salinity-induced CAM, where these parameters are naturally higher, the supplementation of Pip does not yield significant effects. These results validate the hypothesis that the regulatory influence of Pip on defence mechanisms against biotic stress is contingent upon the metabolic state of the plant. Furthermore, this regulatory effect is more pronounced in C3 plants of M. crystallinum than those undergoing CAM metabolism induced by salinity stress.

Does chronic nitrogen deposition have effects on grass physiology of natural habitats?

Anthropogenic activities increase nitrogen deposition, which can have major consequences for biodiversity and ecosystem function. We experimentally investigated how a community of grass species would respond to three levels of nitrogen enrichment, considering their biomass and efficiency of photosynthesis. The experiments were conducted in an open savanna area with sparse trees (cerrado sensu stricto). Fifteen plots were divided considering high nitrogen supplementation, low nitrogen supplementation, and control (without nitrogen application) as treatments. The nitrogen dosages applied in each treatment were based on deposition estimates for 2050 in the Cerrado biome. Three native species had their photosynthetic metabolism evaluated 45 days after the last supplementation. Except for Echinolaena inflexa, all the 16 species found in the area expressed C4 metabolism. Echinolaena inflexa and Loudetiopsis chrysothrix showed an increase in leaf nitrogen with supplementation, with differences in chloroplastidic pigment contents. Differently from L. chrysothrix which increased its chlorophyll content, E. inflexa showed a decrease in chlorophyll and carotenoid contents under N supplementation. E. inflexa is a small species and its leaf expansion under higher contents of nitrogen suggests that this species was affected by shading caused by other community species that increased their biomass. Supplemental nitrogen did not affect the efficiency of photosynthesis of these species or severely change the grass community biomass. However, we noted only a tendency for higher biomass and biomass per tiller for Urochloa decumbens, which suggests a concern about exotic species' performance under conditions of greater availability of nitrogen.

Obituary: Prof. Donovan P Kelly (1940-2024), with a review of his life's work in microbial sulfur metabolism, autotrophy and C1 metabolism

Obituary: Prof. Donovan P Kelly (1940-2024), with a review of his life's work in microbial sulfur metabolism, autotrophy and C1 metabolism

Sistema de silvipastoril com alta densidade de árvores acelera a degradação de capim tropical

ABSTRACT Tree density is an important aspect in silvopastoral system (SPS) planning, since low luminosity can limit forage perenniality, especially for tropical forages of C4 metabolism. The objective with this study was to verify if an SPS with high tree density accelerates the pasture degradation process and changes the forage chemical composition. The experiment was carried out by comparison of marandu palisade grass [Urochloa brizantha (Hochst. ex A. Rich.) R. D. Webster] pasture in two systems: silvopastoral and open pasture. In the SPS, teak (Tectona grandis) was planted with a density of 750 trees ha-1. Evaluations were carried out over three years (2015, 2016 and 2017). SPS shading reduced herbage mass, tiller density and soil cover over the years. In the marandu palisade grass in the SPS there was a greater stem proportion, which favoured lesser potential digestible dry matter in the first year. Even with a higher amount of stem, higher crude protein concentration and minerals were observed in the SPS. Due to the high density of trees, excessive shading accelerated the process of degradation of the pasture, which demonstrates that planning of the spatial arrangement of tree species is crucial.

Identification of FtfL as a novel target of berberine in intestinal bacteria

BackgroundBerberine (BBR) is a commonly used anti-intestinal inflammation drug, and its anti-cancer activity has been found recently. BBR can intervene and control malignant colorectal cancer (CRC) through intestinal microbes, but the direct molecular target and related mechanism are unclear. This study aimed to identify the target of BBR and dissect related mechanisms against the occurrence and development of CRC from the perspective of intestinal microorganisms.ResultsHere, we found that BBR inhibits the growth of several CRC-driving bacteria, especially Peptostreptococcus anaerobius. By using a biotin-conjugated BBR derivative, we identified the protein FtfL (formate tetrahydrofolate ligase), a key enzyme in C1 metabolism, is the molecular target of BBR in P. anaerobius. BBR exhibits strong binding affinity and potent inhibition on FtfL. Based on this, we determined the crystal structure of PaFtfL (P. anaerobius FtfL)-BBR complex and found that BBR can not only interfere with the conformational flexibility of PaFtfL tetramer by wedging the tetramer interface but also compete with its substrate ATP for binding within the active center. In addition, the enzymatic activities of FtfL homologous proteins in human tumor cells can also be inhibited by BBR.ConclusionsIn summary, our study has identified FtfL as a direct target of BBR and uncovered molecular mechanisms involved in the anti-CRC of BBR. BBR interferes with intestinal pathogenic bacteria by targeting FtfLs, suggesting a new means for controlling the occurrence and development of CRC.

Genomic potential and physiological characteristics of C1 metabolism in novel acetogenic bacteria.

Acetogenic bacteria can utilize C1 compounds, such as carbon monoxide (CO), formate, and methanol, via the Wood-Ljungdahl pathway (WLP) to produce biofuels and biochemicals. Two novel acetogenic bacteria of the family Eubacteriaceae ES2 and ES3 were isolated from Eulsukdo, a delta island in South Korea. We conducted whole genome sequencing of the ES strains and comparative genome analysis on the core clusters of WLP with Acetobacterium woodii DSM1030T and Eubacterium limosum ATCC8486T. The methyl-branch cluster included a formate transporter and duplicates or triplicates copies of the fhs gene, which encodes formyl-tetrahydrofolate synthetase. The formate dehydrogenase cluster did not include the hydrogenase gene, which might be replaced by a functional complex with a separate electron bifurcating hydrogenase (HytABCDE). Additionally, duplicated copies of the acsB gene, encoding acetyl-CoA synthase, are located within or close to the carbonyl-branch cluster. The serum bottle culture showed that ES strains can utilize a diverse range of C1 compounds, including CO, formate, and methanol, as well as CO2. Notably, ES2 exhibited remarkable resistance to high concentrations of C1 substrates, such as 100% CO (200 kPa), 700 mM formate, and 500 mM methanol. Moreover, ES2 demonstrated remarkable growth rates under 50% CO (0.45 h-1) and 200 mM formate (0.34 h-1). These growth rates are comparable to or surpassing those previously reported in other acetogenic bacteria. Our study introduces novel acetogenic ES strains and describes their genetic and physiological characteristics, which can be utilized in C1-based biomanufacturing.

MINI-AC: inference of plant gene regulatory networks using bulk or single-cell accessible chromatin profiles.

Gene regulatory networks (GRNs) represent the interactions between transcription factors (TF) and their target genes. Plant GRNs control transcriptional programs involved in growth, development, and stress responses, ultimately affecting diverse agricultural traits. While recent developments in accessible chromatin (AC) profiling technologies make it possible to identify context-specific regulatory DNA, learning the underlying GRNs remains a major challenge. We developed MINI-AC (Motif-Informed Network Inference based on Accessible Chromatin), a method that combines AC data from bulk or single-cell experiments with TF binding site (TFBS) information to learn GRNs in plants. We benchmarked MINI-AC using bulk AC datasets from different Arabidopsis thaliana tissues and showed that it outperforms other methods to identify correct TFBS. In maize, a crop with a complex genome and abundant distal AC regions, MINI-AC successfully inferred leaf GRNs with experimentally confirmed, both proximal and distal, TF-target gene interactions. Furthermore, we showed that both AC regions and footprints are valid alternatives to infer AC-based GRNs with MINI-AC. Finally, we combined MINI-AC predictions from bulk and single-cell AC datasets to identify general and cell-type specific maize leaf regulators. Focusing on C4 metabolism, we identified diverse regulatory interactions in specialized cell types for this photosynthetic pathway. MINI-AC represents a powerful tool for inferring accurate AC-derived GRNs in plants and identifying known and novel candidate regulators, improving our understanding of gene regulation in plants.

Mechanistic insights into sulfur source-driven physiological responses and metabolic reorganization in the fuel-biodesulfurizing Rhodococcus qingshengii IGTS8.

Comparative proteomics and untargeted metabolomics were combined to study the physiological and metabolic adaptations of Rhodococcus qingshengii IGTS8 under biodesulfurization conditions. After growth in a chemically defined medium with either dibenzothiophene (DBT) or MgSO4 as the sulfur source, many differentially produced proteins and metabolites associated with several metabolic and physiological processes were detected including the metabolism of carbohydrates, amino acids, lipids, nucleotides, vitamins, protein synthesis, transcriptional regulation, cell envelope biogenesis, and cell division. Increased production of the redox cofactor mycofactocin and associated proteins was one of the most striking adaptations under biodesulfurization conditions. While most central metabolic enzymes were less abundant in the presence of DBT, a key enzyme of the glyoxylate shunt, isocitrate lyase, was up to 26-fold more abundant. Several C1 metabolism and oligotrophy-related enzymes were significantly more abundant in the biodesulfurizing culture. R. qingshengii IGTS8 exhibited oligotrophic growth in liquid and solid media under carbon starvation. Moreover, the oligotrophic growth was faster on the solid medium in the presence of DBT compared to MgSO4 cultures. In the DBT culture, the cell envelope and phospholipids were remodeled, with lower levels of phosphatidylethanolamine and unsaturated and short-chain fatty acids being the most prominent changes. Biodesulfurization increased the biosynthesis of osmoprotectants (ectoine and mannosylglycerate) as well as glutamate and induced the stringent response. Our findings reveal highly diverse and overlapping stress responses that could protect the biodesulfurizing culture not only from the associated sulfate limitation but also from chemical, oxidative, and osmotic stress, allowing efficient resource management. IMPORTANCE Despite decades of research, a commercially viable bioprocess for fuel desulfurization has not been developed yet. This is mainly due to lack of knowledge of the physiology and metabolism of fuel-biodesulfurizing bacteria. Being a stressful condition, biodesulfurization could provoke several stress responses that are not understood. This is particularly important because a thorough understanding of the microbial stress response is essential for the development of environmentally friendly and industrially efficient microbial biocatalysts. Our comparative systems biology studies provide a mechanistic understanding of the biology of biodesulfurization, which is crucial for informed developments through the rational design of recombinant biodesulfurizers and optimization of the bioprocess conditions. Our findings enhance the understanding of the physiology, metabolism, and stress response not only in biodesulfurizing bacteria but also in rhodococci, a precious group of biotechnologically important bacteria.