Pyruvate Phosphate Dikinase Research Articles

In silico drug target identification and pharmacophore mapping for Leishmania donovani based on metabolic pathways

Leishmania donovani is a protozoan parasite which causes very lethal disease called as visceral leishmaniasis. It is the second killer parasitic disease after malaria. It is transmitted by female sandfly and reported to be spread in 88 countries causes 20,000-30,000 death each year. It is the most neglected tropical disease in terms of drug discovery and development and till now, there is no specific vaccine or drug. We analysed the metabolic pathway of this parasite for identifying potential drug target. The essential node (gene) which is non-homologous to human in the metabolic pathway were considered for network reconstruction. Reconstructed network analysis reveals five drug targets namely: threonine aldolase, Acetyl-CoA acyltransferase, pyruvate orthophosphate dikinase, ATP-binding cassette and P-glycoprotein. These targets are efficient and specific for treating Leishmania donovani parasite. Their pharmacophore is designed and docking studies reveals the action of pharmacophore on these drug targets.

In silico drug target identification and pharmacophore mapping for Leishmania donovani based on metabolic pathways

Leishmania donovani is a protozoan parasite which causes very lethal disease called as visceral leishmaniasis. It is the second killer parasitic disease after malaria. It is transmitted by female sandfly and reported to be spread in 88 countries causes 20,000-30,000 death each year. It is the most neglected tropical disease in terms of drug discovery and development and till now, there is no specific vaccine or drug. We analysed the metabolic pathway of this parasite for identifying potential drug target. The essential node (gene) which is non-homologous to human in the metabolic pathway were considered for network reconstruction. Reconstructed network analysis reveals five drug targets namely: threonine aldolase, Acetyl-CoA acyltransferase, pyruvate orthophosphate dikinase, ATP-binding cassette and P-glycoprotein. These targets are efficient and specific for treating Leishmania donovani parasite. Their pharmacophore is designed and docking studies reveals the action of pharmacophore on these drug targets.

Carbon fixation gene expression in Skeletonema marinoi in nitrogen-, phosphate-, silicate-starvation, and low-temperature stress exposure.

Diatoms are unicellular algae with a set of extraordinary genes, metabolic pathways, and physiological functions acquired by secondary endosymbiosis, especially for their efficient photosynthetic carbon fixation mechanisms, which can be a reason for their successful environmental adaptation and great contribution to primary production. Based on the available genomic information, the expression patterns of carbon fixation genes were analyzed using transcriptomic sequencing and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in Skeletonema marinoi. Meanwhile, suitable reference genes applying to specific experimental treatments were selected. In our results, carbon fixation genes were standardized by actin and TATA box-binding protein-coding genes in growth phase samples and stress conditions, respectively. It was found that a series of carbon fixation genes, such as the pyruvate orthophosphate dikinase (PPDK)-coding gene, had significantly up-regulated expression in nitrogen-starvation, phosphate-starvation, and low-temperature conditions, but consistently down-regulated in silicate-starvation treatment. These carbon fixation genes exhibited variable expression levels in different conditions and will be useful for investigating gene expression mechanisms in S.marinoi and improve our understanding of diatom carbon fixation pathways.

Efficient In Vivo Screening Method for the Identification of C4 Photosynthesis Inhibitors Based on Cell Suspensions of the Single-Cell C4 Plant Bienertia sinuspersici.

The identification of novel herbicides is of crucial importance to modern agriculture. We developed an efficient in vivo assay based on oxygen evolution measurements using suspensions of chlorenchyma cells isolated from the single-cell C4 plant Bienertia sinuspersici to identify and characterize inhibitors of C4 photosynthesis. This novel approach fills the gap between conventional in vitro assays for inhibitors targeting C4 key enzymes and in vivo experiments on whole plants. The assay addresses inhibition of the target enzymes in a plant context thereby taking care of any reduced target inhibition due to metabolization or inadequate uptake of small molecule inhibitors across plant cell walls and membranes. Known small molecule inhibitors targeting C4 photosynthesis were used to validate the approach. To this end, we tested pyruvate phosphate dikinase inhibitor bisindolylmaleimide IV and phosphoenolpyruvate carboxylase inhibitor okanin. Both inhibitors show inhibition of plant photosynthesis at half-maximal inhibitory concentrations in the sub-mM range and confirm their potential to act as a new class of C4 selective inhibitors.

Activities of key enzymes in the C4 pathway and anatomy of sugarcane infected by Leifsonia xyli subsp. xyli.

Ratoon stunting disease caused by Leifsonia xyli subsp. xyli (Lxx) is a bacterial disease that has plagued sugarcane-planting countries for a long time. This study mainly analysed Lxx localization and its effects on sugarcane leaf. Badila were inocultated by bacteria of Lxx. It was noted that the number of Lxx cells were rapidly enriched in sugarcane leaves from the 150th to the 210th days of post inoculation (dpi). Lxx infection disrupted the integrity of vascular bundle sheath cells (BSC) in the 'Kranz anatomy' of leaves, resulting in irregular accumulation of starch in vascular BSC of leaves. In situ PCR showed that the Lxx localized in the xylem vessels, mesophyll cell (MC) and BSC as described before in sugarcane leaf, a new niche within the host tissues in the phloem of sugarcane stem. The gene expression and activities of phosphoenolpyruvate carboxylase (PEPC), pyruvate, orthophosphate dikinase (PPDK) and NADP-malic enzyme (NADP-ME) enzymes were lower in Lxx-inoculated sugarcane plants as compared to the MI group. Lxx infection not only disrupted the structure of vascular BSC in the C4 'Kranz anatomy' of sugarcane leaves, but also affected the activities and gene expression of the key enzymes PEPC, PPDK and NADP-ME in the C4 cycle of sugarcane suggesting a reduction in CO2 fixation. The effect of Leifsonia xyli subsp. xyli (Lxx) infection on the photosynthetic physiology of sugarcane is currently limited to the evaluation of photosynthetic parameters. This study assessed the impact of Lxx infection on the mechanism of C4 cycle CO2 fixation and to accompanying plant anatomy.

Thermodynamic analysis of the pathway for ethanol production from cellobiose in Clostridium thermocellum

Clostridium thermocellum is a candidate for consolidated bioprocessing by carrying out both cellulose solubilization and fermentation. However, despite significant efforts the maximum ethanol titer achieved to date remains below industrially required targets. Several studies have analyzed the impact of increasing ethanol concentration on C. thermocellum’s membrane properties, cofactor pool ratios, and altered enzyme regulation. In this study, we explore the extent to which thermodynamic equilibrium limits maximum ethanol titer. We used the max-min driving force (MDF) algorithm (Noor et al., 2014) to identify the range of allowable metabolite concentrations that maintain a negative free energy change for all reaction steps in the pathway from cellobiose to ethanol. To this end, we used a time-series metabolite concentration dataset to flag five reactions (phosphofructokinase (PFK), fructose bisphosphate aldolase (FBA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH)) which become thermodynamic bottlenecks under high external ethanol concentrations. Thermodynamic analysis was also deployed in a prospective mode to evaluate genetic interventions which can improve pathway thermodynamics by generating minimal set of reactions or elementary flux modes (EFMs) which possess unique genetic variations while ensuring mass and redox balance with ethanol production. MDF evaluation of all generated (336) EFMs indicated that, i) pyruvate phosphate dikinase (PPDK) has a higher pathway MDF than the malate shunt alternative due to limiting CO2 concentrations under physiological conditions, and ii) NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) can alleviate thermodynamic bottlenecks at high ethanol concentrations due to cofactor modification and reduction in ATP generation. The combination of ATP linked phosphofructokinase (PFK-ATP) and NADPH linked alcohol dehydrogenase (ADH-NADPH) with NADPH linked aldehyde dehydrogenase (ALDH-NADPH) or ferredoxin: NADP ​+ ​oxidoreductase (NADPH-FNOR) emerges as the best intervention strategy for ethanol production that balances MDF improvements with ATP generation, and appears to functionally reproduce the pathway employed by the ethanologen Thermoanaerobacterium saccharolyticum. Expanding the list of measured intracellular metabolites and improving the quantification accuracy of measurements was found to improve the fidelity of pathway thermodynamics analysis in C. thermocellum. This study demonstrates even before addressing an organism’s enzyme kinetics and allosteric regulations, pathway thermodynamics can flag pathway bottlenecks and identify testable strategies for enhancing pathway thermodynamic feasibility and function.

The H3K27me3 demethylase REF6 promotes leaf senescence through directly activating major senescence regulatory and functional genes in Arabidopsis.

The roles of histone demethylation in the regulation of plant flowering, disease resistance, rhythmical response, and seed germination have been elucidated recently; however, how histone demethylation affects leaf senescence remains largely unclear. In this study, we exploited yeast one-hybrid (Y1H) to screen for the upstream regulators of NONYELLOWING1 (NYE1), and identified RELATIVE OF EARLY FLOWERING6 (REF6), a histone H3 lysine 27 tri-methylation (H3K27me3) demethylase, as a putative binding protein of NYE1 promoter. By in vivo and in vitro analyses, we demonstrated that REF6 directly binds to the motif CTCGYTY in NYE1/2 promoters through its zinc finger domain and positively regulates their expression. Loss-of-function of REF6 delayed chlorophyll (Chl) degradation, whereas overexpression of REF6 accelerated Chl degradation. Subsequently, we revealed that REF6 positively regulates the general senescence process by directly up-regulating ETHYLENE INSENSITIVE 2 (EIN2), ORESARA1 (ORE1), NAC-LIKE, ACTIVATED BY AP3/PI (NAP), PYRUVATE ORTHOPHOSPHATE DIKINASE (PPDK), PHYTOALEXIN DEFICIENT 4 (PAD4), LIPOXYGENASE 1 (LOX1), NAC DOMAIN CONTAINING PROTEIN 3 (AtNAC3), and NAC TRANSCRIPTION FACTOR-LIKE 9 (NTL9), the key regulatory and functional genes predominantly involved in the regulation of developmental leaf senescence. Importantly, loss-of-function of REF6 increased H3K27me3 levels at all the target Senescence associated genes (SAGs). We therefore conclusively demonstrate that H3K27me3 methylation represents an epigenetic mechanism prohibiting the premature transcriptional activation of key developmentally up-regulated senescence regulatory as well as functional genes in Arabidopsis.

Key changes in gene expression identified for different stages of C4 evolution in Alloteropsis semialata.

C4 photosynthesis is a complex trait that boosts productivity in tropical conditions. Compared with C3 species, the C4 state seems to require numerous novelties, but species comparisons can be confounded by long divergence times. Here, we exploit the photosynthetic diversity that exists within a single species, the grass Alloteropsis semialata, to detect changes in gene expression associated with different photosynthetic phenotypes. Phylogenetically informed comparative transcriptomics show that intermediates with a weak C4 cycle are separated from the C3 phenotype by increases in the expression of 58 genes (0.22% of genes expressed in the leaves), including those encoding just three core C4 enzymes: aspartate aminotransferase, phosphoenolpyruvate carboxykinase, and phosphoenolpyruvate carboxylase. The subsequent transition to full C4 physiology was accompanied by increases in another 15 genes (0.06%), including only the core C4 enzyme pyruvate orthophosphate dikinase. These changes probably created a rudimentary C4 physiology, and isolated populations subsequently improved this emerging C4 physiology, resulting in a patchwork of expression for some C4 accessory genes. Our work shows how C4 assembly in A. semialata happened in incremental steps, each requiring few alterations over the previous step. These create short bridges across adaptive landscapes that probably facilitated the recurrent origins of C4 photosynthesis through a gradual process of evolution.

Genome-wide identification of genes involved in carbon fixation in Saccharina japonica and responses of putative C4-related genes to bicarbonate concentration and light intensity

Brown algae play a dominant role in the primary productivity of coastal ecosystems and may have an efficient carbon fixation. In this work, 56 genes involved in inorganic carbon fixation were identified from the Saccharina japonica genome. Sequence structure analysis of these genes showed the existence of corresponding function domains and active amino acid sites highly conserved with other stramenopile species. The predicted subcellular localizations showed that Calvin cycle-related enzymes predominantly reside in the plastid and that putative C4-related enzymes are mainly distributed in the mitochondrion. We determined the transcriptional profiles and enzymatic activities of these C4-related enzymes in response to the KHCO3 concentrations and light intensities. Pyruvate orthophosphate dikinase (PPDK) presented the greatest response to low HCO3− concentrations and high light intensity. Phosphoenolpyruvate carboxykinase (PEPCK) was up-regulated at low HCO3− concentrations to compensate for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and might be the crucial decarboxylase in this kelp. We propose that S. japonica might possess a PPDK- and PEPCK-dependent C4-like pathway that enables its rapid growth in natural coastal environments.

Proteomic and Phosphoproteomic Analysis in Tobacco Mosaic Virus-Infected Tobacco (Nicotiana tabacum)

Tobacco mosaic virus (TMV) is a common source of biological stress that significantly affects plant growth and development. It is also useful as a model in studies designed to clarify the mechanisms involved in plant viral disease. Plant responses to abiotic stress were recently reported to be regulated by complex mechanisms at the post-translational modification (PTM) level. Protein phosphorylation is one of the most widespread and major PTMs in organisms. Using immobilized metal ion affinity chromatography (IMAC) enrichment, high-pH C18 chromatography fraction, and high-accuracy mass spectrometry (MS), a set of proteins and phosphopeptides in both TMV-infected tobacco and control tobacco were identified. A total of 4905 proteins and 3998 phosphopeptides with 3063 phosphorylation sites were identified. These 3998 phosphopeptides were assigned to 1311 phosphoproteins, as some proteins carried multiple phosphorylation sites. Among them, 530 proteins and 337 phosphopeptides corresponding to 277 phosphoproteins differed between the two groups. There were 43 upregulated phosphoproteins, including phosphoglycerate kinase, pyruvate phosphate dikinase, protein phosphatase 2C, and serine/threonine protein kinase. To the best of our knowledge, this is the first phosphoproteomic analysis of leaves from a tobacco cultivar, K326. The results of this study advance our understanding of tobacco development and TMV action at the protein phosphorylation level.

In silico studies and evaluation of antiparasitic role of a novel pyruvate phosphate dikinase inhibitor in Leishmania donovani infected macrophages

The present work deals with the identification and characterization of a novel inhibitor Z220582104, specific to pyruvate phosphate dikinase, for leishmanicidal activities against free promastigotes and intracellular amastigotes. We have used structure-based drug designing approaches and performed homology modelling, virtual screening and molecular dynamics studies. Primary mouse macrophages and macrophage cell line J774A1 were infected with promastigotes of Leishmania donovani. Both promastigotes and infected macrophages were subjected to treatment with the varying concentrations of Z220582104 or miltefosine for assessment of leishmanicidal activity. The novel inhibitor Z220582104 demonstrated growth inhibitory potential and reduced the viability of the free promastigotes in a concentration- and time-dependent manner. Z220582104 was also effective against the intracellular form of the parasites and reduced the number of amastigotes in macrophages and also lowered the parasite index, compared with the untreated infected macrophages. Although less effective compared with the miltefosine, Z220582104 is well tolerated by the dividing cells and normal human lymphocytes and monocytes with no adverse effects on the growth kinetics or viability. Our in silico and in vitro studies suggested that Leishmania donovani pyruvate phosphate dikinase could be a potential new drug target.

Gluconeogenesis is essential for trypanosome development in the tsetse fly vector.

In the glucose-free environment that is the midgut of the tsetse fly vector, the procyclic form of Trypanosoma brucei primarily uses proline to feed its central carbon and energy metabolism. In these conditions, the parasite needs to produce glucose 6-phosphate (G6P) through gluconeogenesis from metabolism of non-glycolytic carbon source(s). We showed here that two phosphoenolpyruvate-producing enzymes, PEP carboxykinase (PEPCK) and pyruvate phosphate dikinase (PPDK) have a redundant function for the essential gluconeogenesis from proline. Indeed, incorporation of 13C-enriched proline into G6P was abolished in the PEPCK/PPDK null double mutant (Δppdk/Δpepck), but not in the single Δppdk and Δpepck mutant cell lines. The procyclic trypanosome also uses the glycerol conversion pathway to feed gluconeogenesis, since the death of the Δppdk/Δpepck double null mutant in glucose-free conditions is only observed after RNAi-mediated down-regulation of the expression of the glycerol kinase, the first enzyme of the glycerol conversion pathways. Deletion of the gene encoding fructose-1,6-bisphosphatase (Δfbpase), a key gluconeogenic enzyme irreversibly producing fructose 6-phosphate from fructose 1,6-bisphosphate, considerably reduced, but not abolished, incorporation of 13C-enriched proline into G6P. In addition, the Δfbpase cell line is viable in glucose-free conditions, suggesting that an alternative pathway can be used for G6P production in vitro. However, FBPase is essential in vivo, as shown by the incapacity of the Δfbpase null mutant to colonise the fly vector salivary glands, while the parental phenotype is restored in the Δfbpase rescued cell line re-expressing FBPase. The essential role of FBPase for the development of T. brucei in the tsetse was confirmed by taking advantage of an in vitro differentiation assay based on the RNA-binding protein 6 over-expression, in which the procyclic forms differentiate into epimastigote forms but not into mammalian-infective metacyclic parasites. In total, morphology, immunofluorescence and cytometry analyses showed that the differentiation of the epimastigote stages into the metacyclic forms is abolished in the Δfbpase mutant.

Functional Characterization of Cytosolic Pyruvate Phosphate Dikinase Gene (MecyPPDK) and Promoter (MecyPPDKP) of Cassava in Response to Abiotic Stress in Transgenic Tobacco

Pyruvate phosphate dikinase (PPDK) is a key enzyme in the photosynthesis of C4 plants; its expression significantly affects the accumulation of photosynthetic products in C4 plants. Pyruvate phosphate dikinase presents two subtypes, namely, chloroplastic PPDK and cytosolic PPDK. Cytosolic PPDK is ubiquitous in C3 and C4 plants. This subtype is also highly expressed in metabolically active storage organs. Cytosolic PPDK in cassava (Manihot esculenta Crantz, MecyPPDK) is considerately expressed mainly in functional leaves. However, research on the function of PPDK in cassava has not yet been reported. In this study, we cloned the 5′ flanking sequence of MecyPPDK fused to the uidA reporter gene and stably transfected tobacco (Nicotiana benthamiana Domin). Slight β‐glucuronidase activity was observed in transformed tobacco. This activity was upregulated remarkably under low temperature, drought, waterlogging, and abscisic acid treatments. Results suggest that MecyPPDK facilitates the survival of cassava plants in response to abiotic stress. Our results will provide further understanding regarding the regulation of MecyPPDK expression and a novel stress‐inducible promoter in transgenic tobacco.

A Novel Mutation of OsPPDKB, Encoding Pyruvate Orthophosphate Dikinase, Affects Metabolism and Structure of Starch in the Rice Endosperm.

Starch, as a main energy storage substance, plays an important role in plant growth and human life. Despite the fact that several enzymes and regulators involved in starch biosynthesis have been identified, the regulating mechanism of starch synthesis is still unclear. In this study, we isolated a rice floury endosperm mutant M14 from a mutant pool induced by 60Co. Both total starch content and amylose content in M14 seeds significantly decreased, and starch thermal and pasting properties changed. Compound starch granules were defected in the floury endosperm of M14 seeds. Map-based cloning and a complementation test showed that the floury endosperm phenotype was determined by a gene of OsPPDKB, which encodes pyruvate orthophosphate dikinase (PPDK, EC 2.7.9.1). Subcellular localization analysis demonstrated that PPDK was localized in chloroplast and cytoplasm, the chOsPPDKB highly expressed in leaf and leaf sheath, and the cyOsPPDKB constitutively expressed with a high expression in developing endosperm. Moreover, the expression of starch synthesis-related genes was also obviously altered in M14 developing endosperm. The above results indicated that PPDK played an important role in starch metabolism and structure in rice endosperm.

Gluconeogenesis and nitrogen metabolism in maize

Two pathways can be used by gluconeogenesis in plants: one employs phosphoenolpyruvate carboxykinase (PEPCK) and the other pyruvate orthophosphate dikinase (PPDK). The occurrence-location of these enzymes was determined in developing kernels of maize. PPDK was much more abundant than PEPCK in extracts of whole kernels. However, their location within the kernel was different. PPDK was particularly abundant in the peripheral endosperm (in which alanine is abundant), whereas PEPCK was localised in the pedicel and basal endosperm transfer cells (where asparagine is metabolised). The abundance of these enzymes was also determined in maize roots where there was a massive increase in abundance of PEPCK and a small increase in abundance of PPDK when they were fed ammonium; PEPCK was located in the pericycle and various cell types associated with the vasculature. On the other hand, there was a large increase in abundance of PPDK in roots subjected to anoxia (which induces an accumulation of alanine), whereas the abundance of PEPCK was decreased. These results show: firstly, that gluconeogenesis can potentially occur in many different tissues of maize. Secondly, within one organ PPDK can be abundant in some tissues and PEPCK in others. Thirdly, the abundance of PPDK and PEPCK is often associated with the metabolism of certain nitrogenous compounds and can be dramatically altered by factors related to nitrogen metabolism. In maize roots and developing kernels PPDK was associated with alanine metabolism. By contrast, the presence of PEPCK in maize roots and kernels was associated with either ammonium or asparagine metabolism. We propose that gluconeogenesis is often a component of a widespread mechanism that is used in coordinating the import/mobilisation of nitrogenous compounds with their utilisation. Further, potentially component of this mechanism may have provided building blocks that were used in the evolution of processes such as C4 photosynthesis, Crassulacean acid metabolism, stomatal metabolism and the biochemical pH stat.

Characterization of maize leaf pyruvate orthophosphate dikinase using high throughput sequencing.

In C4 photosynthesis, pyruvate orthophosphate dikinase (PPDK) catalyzes the regeneration of phosphoenolpyruvate in the carbon shuttle pathway. Although the biochemical function of PPDK in maize is well characterized, a genetic analysis of PPDK has not been reported. In this study, we use the maize transposable elements Mutator and Ds to generate multiple mutant alleles of PPDK. Loss-of-function mutants are seedling lethal, even when plants were grown under 2% CO2 , and they show very low capacity for CO2 assimilation, indicating C4 photosynthesis is essential in maize. Using RNA-seq and GC-MS technologies, we examined the transcriptional and metabolic responses to a deficiency in PPDK activity. These results indicate loss of PPDK results in downregulation of gene expression of enzymes of the C4 cycle, the Calvin cycle, and components of photochemistry. Furthermore, the loss of PPDK did not change Kranz anatomy, indicating that this metabolic defect in the C4 cycle did not impinge on the morphological differentiation of C4 characters. However, sugar metabolism and nitrogen utilization were altered in the mutants. An interaction between light intensity and genotype was also detected from transcriptome profiling, suggesting altered transcriptional and metabolic responses to environmental and endogenous signals in the PPDK mutants.

A new SNP in cyOsPPDK gene is associated with floury endosperm in Suweon 542.

Pyruvate orthophosphate dikinase (PPDK) is a component of glycolysis to mediate endosperm energy charge by adjusting the ratio of ATP to ADP and AMP that proposed to balance the flow of carbon into starch, protein, fatty acid and amino acid biosynthesis. However, these were inconsistent with the first report of a T-DNA insertional knockout mutant of the rice PPDK gene (flo4) showed that rice with inactivated PPDK gene failed to produce a opaque seeds. Therefore, the PPDK might have multifaceted functions in grain filling stage, which in some ways might depend on the direction of the reversible catalysis. Suweon 542 is a rice (Oryza sativa L.) mutant developed from Oryza sativa ssp. japonica cv. Namil. Suweon 542 has a milky-white floury endosperm suitable for dry filling, with low starch damage, low grain hardness, and fine flour particle size. The mutant locus on chromosome 5 controls the floury endosperm phenotype of Suweon 542. Fine mapping of this locus is required for efficient breeding of rice germplasm suitable for dry milling. In this study, whole genome of Suweon 542 and Milyang 23 were re-sequenced using Illumina HiSeq 2500. Co-segregation analysis of F3:4 family populations derived from Suweon 542/Milyang 23 was performed using eight CAPS markers and phenotypic evaluation of the endosperm. The target region was mapped to a 33kb region and identified to encode cytosolic pyruvate orthophosphate dikinase protein (cyOsPPDK). A G→A SNP in exon 8 of cyOsPPDK resulting in a missense mutation from Gly to Asp at amino acid position 404 was responsible for the floury endosperm of Suweon 542. qRT-PCR experiments revealed that FLO4-4 was expressed to a considerably higher level in Suweon 542 than in Namil during the grain filling stage. Overall, fine mapping of FLO4-4 and candidate gene analysis provided further insight into the floury endosperm of rice, and reveal a novel SNP in cyOsPPDK gene can affect the floury endosperm phenotype through active PPDK gene during grain filling stage.

Changes in nitrogen availability lead to a reprogramming of pyruvate metabolism

BackgroundLow availability of nitrogen (N) severely affects plant growth at different levels, which can be reverted by the resupply of N. To unravel the critical steps in primary metabolism underlying the growth adjustment in response to changes in N availability, transcriptomic and comprehensive metabolite analyses were performed in barley using primary leaves at early and later stages of N deprivation, and after N resupply to N-deficient plants.ResultN deficiency in leaves caused differential regulation of 1947 genes, mostly belonging to the functional classes photosynthesis, cell wall degradation, lipid degradation, amino acid degradation, transcription factors, phytohormone metabolism and receptor-like kinases. Interestingly, 62% of the genes responding to low N were regulated in the opposite direction after two days of N resupply. Reprogramming of gene transcription was linked to metabolic rearrangements and affected the metabolism of amino acids and sugars. The levels of major amino acids, including Glu, Asp, Ser, Gln, Gly, Thr, Ala, and Val, decreased during primary leaf age and, more pronounced, during low N-induced senescence, which was efficiently reverted after resupply of N. A significant decrease was observed for pyruvate and metabolites involved in the TCA cycle under low N, and this was reverted to initial levels after 5 days of N resupply. Correspondingly, transcript levels of genes coding for pyruvate kinase, pyruvate dehydrogenase, and pyruvate orthophosphate dikinase followed the same trend as related metabolites.ConclusionOur results show that upon N limitation a specific pathway for remobilization at the link between glycolysis and TCA cycle in barley is established that is at least partly regulated by a strict reprogramming of the gene coding for pyruvate orthophosphate dikinase. Further analysis of this pathway, its regulatory levels and biochemical changing of pyruvate metabolism enzymes in response to N availability is needed to determine the link between N status and primary metabolism.

Development of a Novel Hygiene Monitoring System Based on the Detection of Total Adenylate (ATP+ADP+AMP)

ATP is the universal energy molecule found in animals, plants, and microorganisms. ATP rapid hygiene monitoring tests have been employed in the food industry to ensure that adequate cleanliness is being maintained. However, because ATP is hydrolyzed to ADP and AMP by metabolic processes, by heat treatment, or under acidic or alkaline conditions, total adenylate (ATP+ADP+AMP [A3]) could be a more reliable sanitation indicator of food residues that may cause biofilm formation and allergen contamination. Therefore, a novel hygiene monitoring system to measure A3 was developed based on the luciferin-luciferase assay with the combination of two enzymes, pyruvate kinase and pyruvate phosphate dikinase, that can convert ADP into ATP and recycle AMP into ATP, respectively. The newly developed A3 assay system afforded stable bioluminescence signals and equivalent linear calibration curves between relative light units (RLU) and the amounts of ATP, ADP, and AMP, respectively. To verify the significance of the A3 method, the ratios of ATP, ADP, and AMP in various food samples were determined; large amounts of ADP and AMP were found in a variety of foods, such as meat, seafood, dairy, nuts, fruits, vegetables, and fermented foods. Sanitation monitoring of stainless steel exposed to raw meat was also examined, and the A3 method achieved a 200-RLU level, the typical benchmark value, after complete washing with detergent and rinsing. In contrast, a conventional ATP method showed less than 200 RLU after only a light cold and hot water rinse. In conclusion, the A3 assay appeared to be suitable for detection of adenylates from food residues that are not detected by the conventional ATP assay.

Seed Dormancy Involves a Transcriptional Program That Supports Early Plastid Functionality during Imbibition.

Red rice fully dormant seeds do not germinate even under favorable germination conditions. In several species, including rice, seed dormancy can be removed by dry-afterripening (warm storage); thus, dormant and non-dormant seeds can be compared for the same genotype. A weedy (red) rice genotype with strong dormancy was used for mRNA expression profiling, by RNA-Seq, of dormant and non-dormant dehulled caryopses (here addressed as seeds) at two temperatures (30 °C and 10 °C) and two durations of incubation in water (8 h and 8 days). Aim of the study was to highlight the differences in the transcriptome of dormant and non-dormant imbibed seeds. Transcript data suggested important differences between these seeds (at least, as inferred by expression-based metabolism reconstruction): dry-afterripening seems to impose a respiratory impairment onto non-dormant seeds, thus glycolysis is deduced to be preferentially directed to alcoholic fermentation in non-dormant seeds but to alanine production in dormant ones; phosphoenolpyruvate carboxykinase, pyruvate phosphate dikinase and alanine aminotransferase pathways appear to have an important gluconeogenetic role associated with the restoration of plastid functions in the dormant seed following imbibition; correspondingly, co-expression analysis pointed out a commitment to guarantee plastid functionality in dormant seeds. At 8 h of imbibition, as inferred by gene expression, dormant seeds appear to preferentially use carbon and nitrogen resources for biosynthetic processes in the plastid, including starch and proanthocyanidins accumulation. Chromatin modification appears to be a possible mechanism involved in the transition from dormancy to germination. Non-dormant seeds show higher expression of genes related to cell wall modification, suggesting they prepare for acrospire/radicle elongation.