ATP-dependent Phosphofructokinase Research Articles

Genome-wide identification of the PFK gene family and their expression analysis in Quercus rubra.

The glycolytic pathway involves phosphofructokinase (PFK), a rate-limiting enzyme that catalyzes the phosphorylation of fructose-6-phosphate. In plants, the two PFK members are ATP-dependent phosphofructokinase (PFK) and pyrophosphate-fructose-6-phosphate phosphotransferase (PFP). However, the functions of the PFK family members in Quercus rubra are not well understood. The purpose of this study was to investigate the genome-wide distribution of the PFK family members and their roles in Q. rubra by performing a systematic study of the phylogenetic relationships, molecular characteristics, motifs, chromosomal and subcellular locations, and cis-elements of QrPFKs. We identified 14 QrPFK genes in the genome of Q. rubra, followed by examining their expression in different tissues, including the roots, stems, and leaves. The phylogenetic tree divided the 14 QrPFK genes into two groups: 11 belonging to PFK and three belonging to PFP. The expression profiles of all 14 proteins were relatively the same in leaves but differed between stems and roots. Four genes (Qurub.02G189400.1, Qurub.02G189400.2, Qurub.09G134300.1, and Qurub.09G134300.2) were expressed at very low levels in both stems and roots, while two (Qurub.05G235500.1 and Qurub.05G235500.1) were expressed at low levels and the others showed relatively high expression in all tissues.

New Insights on Sperm Function in Male Infertility of Unknown Origin: A Multimodal Approach.

The global trend of rising (male) infertility is concerning, and the unidentifiable causes in half of the cases, the so-called unknown origin male infertility (UOMI), demands a better understanding and assessment of both external/internal factors and mechanisms potentially involved. In this work, it was our aim to obtain new insight on UOMI, specifically on idiopathic (ID) and Unexplained male infertility (UMI), relying on a detailed evaluation of the male gamete, including functional, metabolic and proteomic aspects. For this purpose, 1114 semen samples, from males in couples seeking infertility treatment, were collected at the Reproductive Medicine Unit from the Centro Hospitalar e Universitário de Coimbra (CHUC), from July 2018-July 2022. Based on the couples' clinical data, seminal/hormonal analysis, and strict eligibility criteria, samples were categorized in 3 groups, control (CTRL), ID and UMI. Lifestyle factors and anxiety/depression symptoms were assessed via survey. Sperm samples were evaluated functionally, mitochondrially and using proteomics. The results of Assisted Reproduction Techniques were assessed whenever available. According to our results, ID patients presented the worst sperm functional profile, while UMI patients were similar to controls. The proteomic analysis revealed 145 differentially expressed proteins, 8 of which were specifically altered in ID and UMI samples. Acrosin (ACRO) and sperm acrosome membrane-associated protein 4 (SACA4) were downregulated in ID patients while laminin subunit beta-2 (LAMB2), mannose 6-phosphate isomerase (MPI), ATP-dependent 6-phosphofructokinase liver type (PFKAL), STAR domain-containing protein 10 (STA10), serotransferrin (TRFE) and exportin-2 (XPO2) were downregulated in UMI patients. Using random forest analysis, SACA4 and LAMB2 were identified as the sperm proteins with a higher chance of distinguishing ID and UMI patients, and their function and expression variation were in accordance with the functional results. No alterations were observed in terms of lifestyle and psychological factors among the 3 groups. These findings obtained in an experimental setting based on 3 well-defined groups of subjects, might help to validate new biomarkers for unknown origin male infertility (ID and UMI) that, in the future, can be used to improve diagnostics and treatments.

Impacts of river snails rice noodle ingredients addition on the kitchen waste anaerobic digestion performances, microbial communities and metabolic pathways

With the rapid development of the river snails rice noodle industry, the treatment of the wastewater generated in the production of river snails rice noodle has attracted people’s attention. This study investigated the performance of the anaerobic digestion of kitchen waste with river snails rice noodle ingredients, including key enzyme activities, microbial community distribution and metabolic pathways in the anaerobic reactor. The average chemical oxygen demand removal efficiencies were 92.5% and 88.7% when 130 g and 260 g river snails rice noodle ingredients were added in the anaerobic reactor, respectively. Acetate kinase activity of the sludge decreased during anaerobic digestion, but increased at 35 °C on days 55. The river snails rice noodle ingredients inhibited the growth of Proteobacteria and the expression of methane metabolism. An increase in temperature promoted the presence of Bacteroidetes and Methanothrix (increased by 3.95% and 2.64% respectively). The later stage of experiment demonstrated an increase in the glycolysis rate-limiting enzymes ATP-dependent phosphofructokinase and pyruvate kinase. It was illustrated that the addition of river snails rice noodle ingredients inhibited the processing capacity of the anaerobic digestion reactor, and the increase of operating temperature of anaerobic reactor alleviated the inhibition of higher content river snails rice noodle ingredients on anaerobic digestion process. It was provided valuable basic information for the future study of treatment of river snails rice noodle wastewater.

Genome-Wide Analysis, Identification, and Characterization of the PFK Gene Family Members of Populus deltoides

Plants produce two phosphorylated enzymes: pyrophosphate-dependent fructose-6-phosphate phosphotransferase (PFP) and ATP-dependent phosphofructokinase (PFK). However, the characterization of phosphofructokinase (PFK) family members and their functions remains unexplored in Eastern Cottonwood (Populus deltoides). This study comprehensively analyzed 17 phosphofructokinase genes in the P. deltoides genome, including their gene structure, conserved domains, evolutionary relationships, and expression patterns. Genome-wide analysis of the poplar genome identified 11 genes belonging to PdPFK and 6 genes belonging to PdPFP. Phylogenetic, gene structure, and motif analyses revealed two main categories of PFK genes: PFK with eleven genes and PFP with six genes. Quantitative PCR (qPCR) analysis demonstrated that all 17 phosphofructokinase genes were induced in various tissues and developmental stages of P. deltoides. PFPs were strongly expressed throughout P. deltoides’ growth and development, notably in the roots, according to the transcript analysis. This genome-wide analysis provides insights into PFK and PFP genes and establishes a foundation for future investigations into the role of genes in P. deltoids.

The mechanism of Cry41-related toxin against Myzus persicae based on its interaction with Buchnera-derived ATP-dependent 6-phosphofructokinase.

Myzus persicae (Hemiptera: Aphididae) is one of the most notorious pests of many crops worldwide. Most Cry toxins produced by Bacillus thuringiensis show very low toxicity to M. persicae; however, a study showed that Cry41-related toxin had moderate toxic activity against M. persicae. In our previous work, potential Cry41-related toxin-binding proteins in M. persicae were identified, including cathepsin B, calcium-transporting ATPase, and Buchnera-derived ATP-dependent 6-phosphofructokinase (PFKA). Buchnera is an endosymbiont present in almost all aphids and it provides necessary nutrients for aphid growth. This study investigated the role of Buchnera-derived PFKA in Cry41-related toxicity against M. persicae. In this study, recombinant PFKA was expressed and purified, and in vitro assays revealed that PFKA bound to Cry41-related toxin, and Cry41-related toxin at 25 μg ml-1 significantly inhibited the activity of PFKA. In addition, when M. persicae was treated with 30 μg ml-1 of Cry41-related toxin for 24 h, the expression of dnak, a single-copy gene in Buchnera, was significantly decreased, indicating a decrease in the number of Buchnera. Our results suggest that Cry41-related toxin interacts with Buchnera-derived PFKA to inhibit its enzymatic activity and likely impair cell viability, resulting in a decrease in the number of Buchnera, and finally leading to M. persicae death. These findings open up new perspectives in our understanding of the mode of action of Cry toxins and are useful in helping improve Cry toxicity for aphid control. © 2023 Society of Chemical Industry.

Transcriptomics-based Analysis of the Response of Sugar Content in Litchi Pulp to Foliar Calcium Fertilizer Treatment

Ten 16-year-old trees were used as test materials to investigate the effect of foliar calcium fertilizer on the sugar content of ‘Feizixiao’ litchi (Litchi chinensis Sonn.) pulp. The experiment began 35 days after anthesis (DAA) in 2020 and 2021, and the treatment was a foliar spray application of 0.3% CaCl2 aqueous solution, whereas the control was a foliar spray application of water. The sugar content, sucrose-metabolizing enzymes, and ATP-dependent phosphofructokinase (PFK) activities in pulp were measured in 2020 and 2021. Transcriptome sequencing (RNA-seq) was performed on RNA samples from treatment and control fruit pulps at 35, 63, and 69 DAA (full mature stage) in 2020, and 10 genes were chosen for confirmation by real-time polymerase chain reaction (PCR) in 2020 and 2021. At full maturity, the soluble sugar content in the calcium-treated group was extremely significantly or significantly higher than that in the control group. After 63 DAA, the net sucrose-metabolizing enzyme activity in the calcium-treated group was significantly higher than that in the control group. Furthermore, at full maturity, the calcium-treated group had significantly higher sucrose synthase cleavage activity and significantly lower PFK activity than the control group. Fifty-four highly expressed genes in the glycolytic pathway (EMP) were screened from transcriptome data, including hexokinase, PFK, and pyruvate kinase genes; 87% of these genes were downregulated in the treatment group compared with the control group at 69 DAA in 2020. The linear regression between RNA-seq and real-time PCR results was significant in 2020 (r = 0.9292) and 2021 (r = 0.8889). When the fruit is fully ripe, calcium treatment increases net sucrose-metabolizing enzyme activity by increasing sucrose synthase cleavage activity, promoting the accumulation of reducing sugars, and it downregulates phosphofructokinase gene expression in EMP, promoting sugar accumulation.

Decipher the molecular evolution and expression patterns of Cupin family genes in oilseed rape

Cupin proteins are involved in plant growth and development as well as in response to various stresses. Here, a total of 173 Cupin genes were identified in Brassica napus, and their molecular evolution and expression patterns were analyzed. These genes were classified into ten groups. Motif and exon-intron structure indicated a high degree of conservation within each group during evolution. BnaCupins were distributed on 19 chromosomes and their expansion is mainly contributed by whole-genome duplication (WGD) and segmental duplication events. BnaCupins have undergone severe purifying selection during a long evolutionary process. Meanwhile, some positive selection sites were identified. Expression patterns and cis-element analysis indicated that BnaCupins play significant roles in plant growth and stress responses. In addition, the expression levels of some BnCupins were significantly altered when treated with different conditions (cold, salt, drought, IAA, ABA, and 6-BA). Some BnaCupin interacting proteins, such as glycosyl hydrolase5 (GHs5), carbohydrate kinase (CHKs), ATP-dependent 6-phosphofructokinase (ATP-PFK), S-adenosylmethionine synthase (S-MAT), and aldolase class II (ALD II), were identified by the protein-protein interaction network. It will contribute to enriching our knowledge of the Cupin gene family in B. napus and provide a basis for further studies of their functions.

Genome-Wide Identification and Expression Analysis Elucidates the Potential Role of PFK Gene Family in Drought Stress Tolerance and Sugar Metabolism in Cotton.

Drought has been identified as a major threat for global crop production worldwide. Phosphofructokinase (PFK) is vital for sugar metabolism. During phosphorylation, plants have two enzymes: ATP-dependent phosphofructokinase (PFK) and pyrophosphate-dependent fructose-6-phosphate phosphotransferase (PFP). Genome-wide identification led to the identification of 80 PFK genes, 26 genes in G. hirsutum and G. barbadense, and 14 genes in G. arboreum and G. raimondii. Phylogenetic, gene structure, and motif analyses showed that PFK genes were grouped into two main categories, namely, PFK and PFP, with 18 and 8 genes in the allotetraploid species and 10 PFK and 4 PFP genes in the diploid species, respectively. Using the RNA-seq expressions of 26 genes from GhPFK, a co-expression network analysis was performed to identify the hub genes. GhPFK04, GhPFK05, GhPFK09, GhPFK11, GhPFK13, GhPFK14, and GhPFK17 in leaves and GhPFK02, GhPFK09, GhPFK11, GhPFK15, GhPFK16, and GhPFK17 in root tissues were found as hub genes. RT-qPCR analysis validated the expressions of identified hub genes. Interestingly, GhPFK11 and GhPFK17 were identified as common hub genes, and these might be the true candidate genes involved in the drought stress tolerance. In the KEGG enrichment analysis, amino acids such as L-valine, L-histidine, L-glutamine, L-serine, L-homoserine, L-methionine, L-cysteine, and gluconic acid were significantly upregulated, whereas sugars, mainly fructose-1-phosphate, D-mannitol, D-sorbitol, dulcitol, and lactose, were significantly downregulated during drought stress. Genome-wide analysis paves the way for a deeper understanding of the PFK genes and establishes the groundwork for future research into PFK’s role in enhancing drought stress tolerance and sugar metabolism in cotton.

ApoM regulates PFKL through the transcription factor SREBF1 to inhibit the proliferation, migration and metastasis of liver cancer cells.

Apolipoprotein M (ApoM) is considered a protective factor that inhibits the occurrence and development of liver cancer, but the specific underlying mechanisms require further investigation. Previous studies have demonstrated that ApoM gene knockout promotes the expression of the transcription factor sterol regulatory element-binding protein 1 (SREBP1; also known as SREBF1) in the livers of mice. SREBF1 is closely associated with factors involved in fatty acid synthesis and has a role in the promotion of tumor progression. The present study initially confirmed that the expression levels of ApoM in cancer tissues were significantly decreased compared with those in normal tissue, while the expression levels of SREBF1 were significantly increased. In addition, ApoM gene knockout significantly increased the expression levels of SREBF1 and the key glycolytic enzyme ATP-dependent 6-phosphofructokinase, liver type (PFKL). Binding site prediction and a dual-luciferase reporter gene assay indicated that SREBF1 regulates the promoter region of PFKL. To the best of our knowledge, the present study was the first to propose the regulation of glycolytic enzyme transcription levels by SREBF1. Furthermore, cell proliferation and Transwell assays demonstrated that ApoM gene knockout increased the expression levels of SREBF1 and further enhanced the activity of the promoter region of PFKL, ultimately promoting the proliferation, migration and invasion of liver cancer cells.

Osteoblasts induce glucose-derived ATP perturbations in chondrocytes through noncontact communication.

Cartilage and subchondral bone communicate with each other through material and signal exchanges. However, direct evidence provided by experimental studies on their interactions is insufficient. In the present study, we establish a noncontact co-culture model with a transwell chamber to explore the energetic perturbations in chondrocytes influenced by osteoblasts. Our results indicate that osteoblasts induce more ATP generation in chondrocytes through an energetic shift characterized by enhanced glycolysis and impaired mitochondrial tricarboxylic acid cycle. Enhanced glycolysis is shown by an increase of secreted lactate and the upregulation of glycolytic enzymes, including glucose-6-phosphate isomerase (Gpi), liver type ATP-dependent 6-phosphofructokinase (Pfkl), fructose-bisphosphate aldolase C (Aldoc), glyceraldehyde-3-phosphate dehydrogenase (Gapdh), triosephosphate isomerase (Tpi1), and phosphoglycerate kinase 1 (Pgk1). Impaired mitochondrial tricarboxylic acid cycle is characterized by the downregulation of cytoplasmic aspartate aminotransferase (Got1) and mitochondrial citrate synthase (Cs). Osteoblasts induce the activation of Akt and P38 signaling to mediate ATP perturbations in chondrocytes. This study may deepen our understanding of the maintenance of metabolic homeostasis in the bone-cartilage unit.

Dynamics of Energy Metabolism in Carbon Starvation-Induced Fruitlet Abscission in Litchi

Fruit abscission is triggered by multiple changes in endogenous components of the fruit, including energy metabolism. However, it is still unknown how the core energy metabolism pathways are modified during fruit abscission. Here, we investigated the relationship between carbon starvation-induced fruitlet abscission and energy metabolism changes in litchi. The fruitlet abscission of litchi ‘Feizixiao’ was induced sharply by girdling plus defoliation (GPD), a carbon stress treatment. Using liquid chromatography tandem mass spectrometry (LC-MS/MS) targeted metabolomics analysis, we identified a total of 21 metabolites involved in glycolysis, TCA cycle and oxidative phosphorylation pathways. Among them, the content of most metabolites in glycolysis pathways and TCA cycles was reduced, and the activity of corresponding metabolic enzymes such as ATP-dependent phosphofructokinase (ATP-PFK), pyruvate kinase (PK), citrate synthase (CS), succinate thiokinase (SAT), and NAD-dependent malate dehydrogenase (NAD-MDH) was decreased. Consistently, we further showed that the expression of the relative genes (LcPFK2, LcPK2, LcPK4, LcCS1, LcCS2, LcSAT, LcMDH1 and LcMDH2) was also significantly down-regulated. In contrast, the level of ATP, an important metabolite in the oxidative phosphorylation pathway, was elevated in parallel with both higher activity of H+-ATPase and the increased expression level of LcH+-ATPase1. In conclusion, our findings suggest that carbon starvation can induce fruitlet abscission in litchi probably by energy depletion that mediated through both the suppression of the glycolysis pathway and TCA cycle and the enhancement of the oxidative phosphorylation pathway.

Identification of three gene families coordinating the conversion between fructose-6-phosphate and fructose-1,6-bisphosphate in wheat

Saccharides are a direct energy source for most organisms and the primary components in grains of common wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD). However, genes involved in the metabolism of primary saccharides such as glucose and fructose have not been fully characterized in wheat, which limits our understanding of how these genes influence wheat growth. In this study, genes coding ATP-dependent phosphofructokinase (PFK), fructose-1,6-bisphosphatase (FBP), and pyrophosphate-dependent fructose-6-phosphate 1-phosphotransferase (PFP), which participate in the conversion between fructose 6-phosphate (F-6-P) and fructose 1,6-bisphosphate (F-1,6-P2), were identified at the genome-wide level. A total of 24, 13, and 12 genes were found encoding TaPFK, TaFBP, and TaPFP, respectively. All predicted peptides of these genes exhibited conserved substrate-binding domain, suggesting they are active enzymes in vivo. Transcriptome data ranked the gene levels as follows: TacyFBP-1 > TacpFBP-1 > TaPFPα-2 ≈ TaPFPβ >> TaPFK-1 ≈ TaPFK-5 >> all remaining genes at different developmental stages of wheat. In the three tapfp-a, b, and d knockout lines, there was a decrease in the plant height, anther length, and thousand-grain mass, while the percentage of abnormal pollen increased compared to that of wild type cv. Huapei3 (HP3). During germination, tapfpβ-a exhibited a lower germination rate, shorter coleoptile and primary root length, and higher fructose content than HP3, tapfpβ-b, and tapfpβ-d lines. Expressions were ranked as follows: TaPFK-5 ≈ TaPFPα-2 >> TaPFPα-1 ≈ TaPFPβ > TacyFBP-1 ≈ TaPFK-7, 9 in HP3. All these genes were downregulated during the 24 - 96 h germinating process in three mutant lines. Collectively, main TaPFK, TaFBP, and TaPFP members cooperated during wheat growth, while TaPFPβ knockout decreased wheat vitality. Results from this study can aid more systematic studies of the physiological and molecular functions of TaPFK, TaFBP, and TaPFP.

Carbon sources driven supernatant micro-particles differentiate in submerged anaerobic membrane bioreactors (AnMBRs)

Recently, micro-particles with a size range of 0.45–10 μm in anaerobic membrane bioreactors (AnMBRs), were rich in microorganisms and characterized as a significant source to membrane fouling. Nevertheless, little is known about the formation of the micro-particles associated microorganisms as well as their metabolic functions, which could be largely impacted by carbon sources in the influent. Here, protein and glucose were chosen to be two different carbon sources fed into two identical AnMBRs (i.e., AnMBR-P and AnMBR-G), and a comprehensive comparison of the micro-particles formed in corresponding reactors was made. Size-dependent fractionation and fluorescent staining assay showed that micro-particles with the size range of 0.45–1 μm possessed the highest contents (41.4%) in the AnMBR-P and presented as vibrios and coccus, while the 5–10 μm micro-particles with filamentous morphologies were the prevailing fraction (61.9%) in the AnMBR-G. According to the results of high-throughput 16S rRNA gene sequencing, the Lentimicrobiaceae, D8A-2 and Syntrophorhabdus prevailed in the AnMBR-P micro-particles, while the SJA-15 showed overwhelming predominance than the other genera in the AnMBR-G micro-particles. Metagenomic sequencing further revealed that the micro-particles in the AnMBR-P possessed higher abundance in specific functional genes for nitrogen metabolism (nirB), sulfur metabolism (cysK) and cysteine and methionine metabolism (mtaD), as well as in the cell mobility as chemotaxis (rbsB) and flagellar assembly (fliC). In comparison, the micro-particles obtained from the AnMBR-G encoded abundant genes for converting glucose to metabolites in the next level, such as glk (glucokinase), pfk (ATP-dependent phosphofructokinase), and PGK (phosphoglycerate kinase). Overall, our findings provided new insights into the formation of micro-particles in the supernatant of AnMBRs, which could facilitate to propose related fouling alleviation methods from the perspective of carbon sources regulation and/or pretreatment of the influent.

Genetic Dissection of Seedling Root System Architectural Traits in a Diverse Panel of Hexaploid Wheat through Multi-Locus Genome-Wide Association Mapping for Improving Drought Tolerance.

Cultivars with efficient root systems play a major role in enhancing resource use efficiency, particularly water absorption, and thus in drought tolerance. In this study, a diverse wheat association panel of 136 wheat accessions including mini core subset was genotyped using Axiom 35k Breeders’ Array to identify genomic regions associated with seedling stage root architecture and shoot traits using multi-locus genome-wide association studies (ML-GWAS). The association panel revealed a wide variation of 1.5- to 50-fold and were grouped into six clusters based on 15 traits. Six different ML-GWAS models revealed 456 significant quantitative trait nucleotides (QTNs) for various traits with phenotypic variance in the range of 0.12–38.60%. Of these, 87 QTNs were repeatedly detected by two or more models and were considered reliable genomic regions for the respective traits. Among these QTNs, eleven were associated with average diameter and nine each for second order lateral root number (SOLRN), root volume (RV) and root length density (RLD). A total of eleven genomic regions were pleiotropic and each controlled two or three traits. Some important candidate genes such as Formin homology 1, Ubiquitin-like domain superfamily and ATP-dependent 6-phosphofructokinase were identified from the associated genomic regions. The genomic regions/genes identified in this study could potentially be targeted for improving root traits and drought tolerance in wheat.

S. aureus Biofilm Protein Expression Linked to Antimicrobial Resistance: A Proteomic Study.

Simple SummaryBiofilm formation represents one of the most effective forms of bacterial persistence in surfaces where nutrients are available or in the tissues of living hosts as humans or animals. Such persistence is due to the high rate of antimicrobial resistance of this shell conformation. It often represents a burden when the pathogen colonizes niches from where it is not removable such as food facilities, farm facilities or parts of living organisms. In this study, we investigated biofilm formation mechanisms and enhanced antimicrobial resistance of 6 different S. aureus strains. The detected mechanisms were primarily related to the control of catabolites, the production of proteins with moonlighting activities and the detoxification of compounds with antimicrobial activities (i.e., alcohol). Glycolysis and aerobic metabolisms were found to be less active in the biofilm conformation. Consequently, less H2O2 production from aerobic metabolism was translated into a measurable under-representation of catalase protein.Antimicrobial resistance (AMR) represents one of the most critical challenges that humanity will face in the following years. In this context, a “One Health” approach with an integrated multidisciplinary effort involving humans, animals and their surrounding environment is needed to tackle the spread of AMR. One of the most common ways for bacteria to live is to adhere to surfaces and form biofilms. Staphylococcus aureus (S. aureus) can form biofilm on most surfaces and in a wide heterogeneity of environmental conditions. The biofilm guarantees the survival of the S. aureus in harsh environmental conditions and represents an issue for the food industry and animal production. The identification and characterization of biofilm-related proteins may provide interesting insights into biofilm formation mechanisms in S. aureus. In this regard, the aims of this study were: (i) to use proteomics to compare proteomes of S. aureus growing in planktonic and biofilm forms in order to investigate the common features of biofilm formation properties of different strains; (ii) to identify specific biofilm mechanisms that may be involved in AMR. The proteomic analysis showed 14 differentially expressed proteins among biofilm and planktonic forms of S. aureus. Moreover, three proteins, such as alcohol dehydrogenase, ATP-dependent 6-phosphofructokinase, and fructose-bisphosphate aldolase, were only differentially expressed in strains classified as high biofilm producers. Differentially regulated catabolites metabolisms and the switch to lower oxygen-related metabolisms were related to the sessile conformation analyzed.

γ-Aminobutyric Acid Enhances Heat Tolerance Associated with the Change of Proteomic Profiling in Creeping Bentgrass.

γ-Aminobutyric acid (GABA) participates in the regulation of adaptability to abiotic stress in plants. The objectives of this study were to investigate the effects of GABA priming on improving thermotolerance in creeping bentgrass (Agrostis stolonifera) based on analyses of physiology and proteome using iTRAQ technology. GABA-treated plants maintained significantly higher endogenous GABA content, photochemical efficiency, performance index on absorption basis, membrane stability, and osmotic adjustment (OA) than untreated plants during a prolonged period of heat stress (18 days), which indicated beneficial effects of GABA on alleviating heat damage. Protein profiles showed that plants were able to regulate some common metabolic processes including porphyrin and chlorophyll metabolism, glutathione metabolism, pyruvate metabolism, carbon fixation, and amino acid metabolism for heat acclimation. It is noteworthy that the GABA application particularly regulated arachidonic acid metabolism and phenylpropanoid biosynthesis related to better thermotolerance. In response to heat stress, the GABA priming significantly increased the abundances of Cu/ZnSOD and APX4 that were consistent with superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities. The GABA-upregulated proteins in relation to antioxidant defense (Cu/ZnSOD and APX4) for the reactive oxygen species scavenging, heat shock response (HSP90, HSP70, and HSP16.9) for preventing denatured proteins aggregation, stabilizing abnormal proteins, promoting protein maturation and assembly, sugars, and amino acids metabolism (PFK5, ATP-dependent 6-phosphofructokinase 5; FK2, fructokinase 2; BFRUCT, β-fructofuranosidase; RFS2, galactinol-sucrose galactosyltransferase 2; ASN2, asparagine synthetase 2) for OA and energy metabolism, and transcription factor (C2H2 ZNF, C2H2 zinc-finger protein) for the activation of stress-defensive genes could play vital roles in establishing thermotolerance. Current findings provide an illuminating insight into the new function of GABA on enhancing adaptability to heat stress in plants.

Short communication: Effect of supplemental zinc source with and without evaporative cooling on systemic and mammary metabolism of lactating dairy cows during summer

The negative effects of heat stress partly result from disturbed systemic metabolic responses and possibly altered mammary gland metabolism of lactating dairy cows. Our previous research reported that supplemental dietary Zn sources may affect milk fat synthesis of lactating cows during summer. Thus, our objective was to evaluate the systemic and mammary metabolism of cows fed 2 supplemental Zn sources under 2 environmental conditions. Multiparous lactating Holstein cows (n = 72; days in milk: 99.7 ± 13.4 d; parity: 2.9 ± 0.3) were randomly assigned to 4 treatments in a 2 × 2 factorial arrangement. Treatments included 2 different environments: cooled (CL) using fans and misters or noncooled (NC), and 2 supplemental Zn sources: 75 mg of Zn hydroxychloride/kg of DM (IOZ) or 35 mg of Zn hydroxychloride/kg of DM + 40 mg of Zn-Met complex/kg of DM (ZMC). The 168-d experiment was divided into baseline and environmental challenge phases, 84 d each. During the baseline phase, all cows were cooled and fed respective dietary treatments, and during the environmental challenge phase cows continued receiving the same diets but NC cows were deprived of cooling. Temperature-humidity index averaged 77.6 ± 3.8 and 77.8 ± 3.8 for CL and NC pens, respectively, during the environmental challenge phase. Plasma was collected before the baseline phase and at 1, 3, 5, 12, 22, 26, 41, 54, 61, 68, 75, and 81 d of the environmental challenge phase for metabolites and insulin analyses. Mammary biopsies were collected before the baseline phase and at 7 and 56 d of the environmental challenge phase to measure mRNA abundance of proteins related to mammary metabolism. Compared with CL, NC reduced plasma glucose, nonesterified fatty acids, β-hydroxybutyrate, and triglyceride concentrations, but increased insulin concentration. Cows fed ZMC had greater plasma triglyceride concentration than IOZ. Treatments had no effect on mRNA abundance of protein related to mammary fatty acid and glucose metabolism except that NC cows had greater mammary mRNA abundance of 6-phosphogluconate dehydrogenase and ATP-dependent 6-phosphofructokinase than CL cows. In conclusion, deprivation of evaporative cooling influenced the metabolism of lactating dairy cows but dietary Zn source had no apparent effect.

Metabolic role of pyrophosphate-linked phosphofructokinase pfk for C1 assimilation in Methylotuvimicrobium alcaliphilum 20Z

BackgroundMethanotrophs is a promising biocatalyst in biotechnological applications with their ability to utilize single carbon (C1) feedstock to produce high-value compounds. Understanding the behavior of biological networks of methanotrophic bacteria in different parameters is vital to systems biology and metabolic engineering. Interestingly, methanotrophic bacteria possess the pyrophosphate-dependent 6-phosphofructokinase (PPi-PFK) instead of the ATP-dependent 6-phosphofructokinase, indicating their potentials to serve as promising model for investigation the role of inorganic pyrophosphate (PPi) and PPi-dependent glycolysis in bacteria. Gene knockout experiments along with global-omics approaches can be used for studying gene functions as well as unraveling regulatory networks that rely on the gene product.ResultsIn this study, we performed gene knockout and RNA-seq experiments in Methylotuvimicrobium alcaliphilum 20Z to investigate the functional roles of PPi-PFK in C1 metabolism when cells were grown on methane and methanol, highlighting its metabolic importance in C1 assimilation in M. alcaliphilum 20Z. We further conducted adaptive laboratory evolution (ALE) to investigate regulatory architecture in pfk knockout strain. Whole-genome resequencing and RNA-seq approaches were performed to characterize the genetic and metabolic responses of adaptation to pfk knockout. A number of mutations, as well as gene expression profiles, were identified in pfk ALE strain to overcome insufficient C1 assimilation pathway which limits the growth in the unevolved strain.ConclusionsThis study first revealed the regulatory roles of PPi-PFK on C1 metabolism and then provided novel insights into mechanism of adaptation to the loss of this major metabolic enzyme as well as an improved basis for future strain design in type I methanotrophs.

Genome-wide identification, expression and functional analysis of the phosphofructokinase gene family in Chinese white pear (Pyrus bretschneideri)

Phosphofructokinase plays an essential role in sugar metabolism in plants. Plants possess two types of phosphofructokinase proteins for phosphorylation of fructose-6-phosphate, the pyrophosphate-dependent fructose-6-phosphate phosphotransferase (PFP), and the ATP-dependent phosphofructokinase (PFK). Until now, the gene evolution, expression patterns, and functions of phosphofructokinase proteins were unknown in pear. In this report, 14 phosphofructokinase genes were identified in pear. The phylogenetic tree indicated that the phosphofructokinase gene family could be grouped into two subfamilies, with 10 genes belonging to the PbPFK subfamily, and 4 genes belonging to the PbPFP subfamily. Conserved motifs and exon numbers of the phosphofructokinase were found in pear and other six species. The evolution analysis indicated that WGD/Segmental and dispersed duplications were the main duplication models for the phosphofructokinase genes expansion in pear and other six species. Analysis of cis-regulatory element sequences of all phosphofructokinase genes identified light regulation and the MYB binding site in the promoter of all pear phosphofructokinase genes, suggesting that phosphofructokinase might could be regulated by light and MYB transcription factors (TFs). Gene expression patterns revealed that PbPFP1 showed similar pattern with sorbitol contents, suggesting important contributions to sugar accumulation during fruit development. Further functional analysis indicated that the phosphofructokinase gene PbPFP1 was localized on plasma membrane compartment, indicating that PbPFP1 had function in plasma membrane. Transient transformation of PbPFP1 in pear fruits led to significant increases of fructose and sorbitol compared to controls. Overall, our study provides important insights into the gene expression patterns and important potential functions of phosphofructokinase for sugar accumulation in pear fruits, which will help to enrich understanding of sugar-related bio-pathways and lay the molecular basis for fruit quality improvement.

The kinetic characteristics of human and trypanosomatid phosphofructokinases for the reverse reaction.

Eukaryotic ATP-dependent phosphofructokinases (PFKs) are often considered unidirectional enzymes catalysing the transfer of a phospho moiety from ATP to fructose 6-phosphate to produce ADP and fructose 1,6-bisphosphate. The reverse reaction is not generally considered to occur under normal conditions and has never been demonstrated for any eukaryotic ATP-dependent PFKs, though it does occur in inorganic pyrophosphate-dependent PFKs and has been experimentally shown for bacterial ATP-dependent PFKs. The evidence is provided via two orthogonal assays that all three human PFK isoforms can catalyse the reverse reaction in vitro, allowing determination of kinetic properties. Additionally, the reverse reaction was shown possible for PFKs from three clinically important trypanosomatids; these enzymes are contained within glycosomes in vivo. This compartmentalisation may facilitate reversal, given the potential for trypanosomatids to have an altered ATP/ADP ratio in glycosomes compared with the cytosol. The kinetic properties of each trypanosomatid PFK were determined, including the response to natural and artificial modulators of enzyme activity. The possible physiological relevance of the reverse reaction in trypanosomatid and human PFKs is discussed.